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Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
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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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A critical role for costimulation in negative selection has been
reported in studies that demonstrated a defect in negative selection in
CD40 ligand
(CD40L)2-deficient
mice (3, 4, 5). However, the mechanism by which CD40L
functions in negative selection has not yet been elucidated.
Stimulation of CD40 on APCs by engagement of CD40L expressed on T cells
results in increased levels of cell surface molecules including B7-1,
B7-2, MHC-II, and LFA-1, as well as increased production of a number of
cytokines such as IL-12 and TNF-
(reviewed in Ref. 6).
Recently, however, it has been shown that signaling during CD40-CD40L
interactions may be bidirectional (7). Signaling through
CD40L on T cells has been implicated in promoting cytokine expression
and short term T cell effector responses (8). Blair et al.
(9) have found that activation of peripheral
CD4+ T cells through engagement of CD3 and CD40L
results in significantly more apoptotic death than is induced by
anti-CD3/CD28 treatment. Thus, signaling through CD40L appears to
facilitate activation (and death) of mature T cells. This led us to
consider the possibility that direct signaling through CD40L might also
play a role in thymic negative selection.
To address the question of whether CD40L functions in a cell-autonomous manner during negative selection, we have used bone marrow chimeras in which developing thymocytes either do or do not express CD40L. We conclude that CD40L knockout (KO) thymocytes are negatively selected as efficiently as CD40L wild-type (WT) thymocytes when these populations coexist in a chimeric environment. Thus, CD40-CD40L interactions can function in negative selection through a non-cell-autonomous mechanism that does not require signaling through CD40L specifically on those T cells undergoing negative selection. Finally, although the absence of CD40-CD40L interactions clearly impairs negative selection of autoreactive CD4+ and CD8+ cells during thymic development, we find that self-reactive CD4+ T cells but not CD8+ T cells are eliminated at a late stage of development through a CD40L-independent pathway, resulting in diminished levels of these cells in the mature CD4+ T cell population.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-CD4, -CD8, -CD45.1 (Ly5.2), and -TCR V
Abs were
purchased from PharMingen (San Diego, CA). TCR V Abs used were as
follows: KJ25, anti-murine V3; MR9-4, anti-murine V5.1,
V5.2; RR4-7, anti-murine V6; MR5-2, anti-murine V8.1,
V8.2; V21.5, anti-murine V10, RR3-15, anti-murine V11;
MR11-1, anti-murine V12; and H57-597, anti-murine TCR
-chain.
Mice
BALB/c (BALB), C57BL/6 (B6), and BALB/c x C57BL/6 F1 (CB6F1) mice were obtained from Frederick Cancer Research Facility (Frederick, MD) and were maintained at Bioqual (Rockville, MD). To generate I-E+ CD40L WT and CD40L KO mice expressing the desired superantigens (SAgs), female CD40L KO mice on a B6 background (mammary tumor virus (Mtv)-8+, -9+, and -I-E-) were crossed with CBA/J males (Mtv-6+, -7+, -8+, -9+, -17+, and -I-E+). The CD40L gene is carried on the X chromosome, and male progeny from this cross are therefore B6 x CBA/J CD40L KO. To generate CD40L WT male mice, CBA/J (CD40L WT) females were crossed with B6 (CD40L WT) males. B6 CD40L KO, B6 CD28 KO, BALB CD28 KO, and BALB CD40 KO mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were maintained at Bioqual. BALB mice deficient in both B7-1-deficient and B7-2 (BALB B7 double-KO (DKO)) were a generous gift from A. Sharpe (10). CB6F1 CD28+/- and CB6F1 CD28-/- mice were generated by crossing B6 CD28+/+ x B6 CD28-/- mice and then crossing B6 CD28+/- progeny to BALB CD28-/- mice.
Flow cytometric analysis
Single-cell suspensions were prepared from thymus and spleen.
Anti-FcR mAb 24G2 (blocks Fc
II and FcIII) was added to prevent
FcR-mediated binding of mAb to cells, and cells were then incubated
with FITC-labeled mAb, PE-labeled mAb, biotinylated mAb, and Cy5
conjugate (PharMingen) sequentially. Viable cells were analyzed by
FACScan (BD Biosciences, San Jose, CA) using CellQuest software. When
necessary, four-color staining was performed using FITC-labeled mAb,
PE-labeled mAb, Cy-5-labeled mAb biotinylated-mAb, and
streptavidin-Alexa 594 conjugate (Molecular Probes, Eugene, OR). Viable
cells, as determined by propidium iodide exclusion, were then
analyzed on a dual-laser FACStarPlus (BD
Biosciences). The percent of cells expressing a particular V was
determined as follows: (% V+ - % isotype
control+)/(% H57 (TCR)+
- % isotype control+).
Mixed bone marrow chimeras
Radiation bone marrow chimeras were prepared as described previously (11). Recipient mice were lethally irradiated with 1000 rad and reconstituted with 107 T cell-depleted bone marrow cells. CD40L WT/CD40L KO mixed chimeras were generated by combining equal numbers of bone marrow cells from B6.Ly5.1 x CBA/J (CD40L WT) and from B6.Ly5.2 x CBA/J (CD40L KO) males and reconstituting lethally irradiated B6.Ly5.1 x CBA/J (CD40L WT) host mice. CD40L KO-only chimeras were generated by reconstituting B6.Ly5.1 x CBA/J (CD40L KO) host mice with bone marrow from B6.Ly5.2 x CBA/J (CD40L KO) males. Analysis of chimeras was performed 6–10 wk after reconstitution.
Statistical analyses
Statistical differences were established by Student t test. Values given were obtained by two-tailed paired t tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have examined the role of CD40L in a SAg-mediated model of
negative selection in which thymocytes expressing TCRs that recognize
endogenous retroviral Ags associated with permissive MHC class II (I-E)
molecules are deleted during thymic development (reviewed in Ref.
12). Female B6 CD40L WT or CD40L KO mice were crossed with
male CBA/J mice to generate male B6 x CBA/J
(Mtv-6+, -7+,
-8+, -9+,
-17+, and -I-E+) offspring
that were CD40L WT or CD40L KO, respectively. Negative selection in
response to Mtv-7, -8, and -9 was then examined in these mice. As
illustrated in Fig. 1
, CD4+ single-positive (SP) thymocytes expressing
V5, V6, V11, and V12 were almost undetectable in the
B6 x CBA/J CD40L WT mice but were clearly present in the B6
x CBA/J CD40L KO mice, consistent with earlier reports that negative
selection of self-reactive T cells is impaired in CD40L-deficient mice
(3, 4, 5). A similar pattern of defective negative selection
was observed in the CD8+ SP population of B6
x CBA/J CD40 KO mice (data not shown). The frequency of
V10-expressing thymocytes was measured as a control for the
specificity of deletion, given that V10-containing TCRs do not
recognize endogenous Mtv products. In fact, a compensatory increase in
the proportion of V10-expressing cells was observed in CD40L WT
relative to CD40L KO mice.
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5, V6, V11, and
V12-expressing thymocytes was then assessed in both CD40L WT and
CD40L KO thymocytes. Our expectation was that if negative selection of
a thymic T cell requires signaling through CD40L expressed by that T
cell, then only those thymocytes derived from the CD40L WT donor would
be deleted in response to SAgs in the chimeras. If, however, the role
of CD40L during negative selection is not cell autonomous, then
deletion should proceed in both CD40L KO and WT donors. CD40L WT/CD40L
KO chimeras were generated by combining equal numbers of bone marrow
cells from B6 x CBA/J CD40L WT and B6 x CBA/J CD40L KO
males and reconstituting lethally irradiated B6 x CBA/J CD40L WT
host mice. The B6 x CBA/J CD40L KO donor cells were
Ly5.2+ and thus could be distinguished from the
B6 x CBA/J CD40L WT Ly5.2- donor and host
populations. Staining for Ly5.2 on thymocytes indicated that CD40L WT
and CD40L KO donor-derived cells were present in approximately equal
numbers in the thymus (Ly5.2- (CD40L WT),
56 ± 3%; Ly5.2+ (CD40L KO), 44 ±
3%) and was similar in all thymic subpopulations (data not shown).
Furthermore, when splenocytes from the chimeras were stimulated with
PMA-ionomycin, CD40L was up-regulated on Ly5.2-
but not on Ly5.2+ T cells, confirming the ability
of Ly5.2 expression to distinguish CD40L-expressing and -deficient T
cell populations in these chimeras (Fig. 2). Analysis of V expression in the
CD40L WT/CD40L KO chimeras revealed that, as expected, deletion of
V5-, V6-, V11-, and V12-expressing thymocytes occurred in
the CD40L WT population (Fig. 3). Of
particular interest was the fate of V5-, V6-, V11-, and
V12-expressing thymocytes in the CD40L KO population. We found that,
similar to the CD40L WT population, CD4+ and
CD8+ SP thymocytes expressing V5, V6,
V11, and V12 were deleted in the CD40L KO population. In
contrast, in CD40L KO-only chimeras, made by reconstituting CD40L KO
host mice with exclusively CD40L KO bone marrow, deletion of V5-,
V6-, V11 and V12-expressing thymocytes was impaired. In CD4 SP
thymocytes, the levels of V5-, V6-, V11-, and
V12-expressing thymocytes in the CD40L KO-only chimera were similar
to those found in the I-E-, nondeleting C57BL/6
controls. In CD8 SP thymocytes, the comparability with the C57BL/6
control was less consistent. Overall, the results from the CD40L
WT/CD40L KO chimeras indicate that direct cell-autonomous signaling via
CD40L is not required for SAg-mediated negative selection.
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The results from the CD40L WT/CD40L KO chimeras suggest that the
role of CD40L during negative selection may be to regulate expression
of other costimulatory molecules and/or cytokines through engagement of
CD40, the only known CD40LR. To test the role of CD40 directly,
SAg-mediated negative selection was examined in BALB CD40 KO mice. BALB
mice are I-E+; express Mtv Ags 6, 8, and 9; and
delete thymocytes expressing a number of Vs including V5, V11,
and V12. Comparison of V5, V11, and V12 frequencies in
thymocytes from B6 (nondeleting strain), BALB CD40 WT, and BALB CD40 KO
controls revealed impaired negative selection in CD40 KO mice (Fig. 4) similar to the defect observed in
CD40L KO mice. These findings indicate that interaction of CD40 with
CD40L is required for intrathymic negative selection.
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5-, V11-, and V12-expressing thymocytes was
similar in CB6F1 (I-E+,
Mtv-6+, Mtv-8+,
Mtv-9+) CD28 KO and CB6F1
CD28+/- mice (Fig. 5). SAg-mediated negative selection in
BALB B7 DKO mice was assessed to determine whether the failure to
observe effects on negative selection in CD28 KO animals might reflect
the use of alternate B7 receptors such as CTLA-4. However, as
shown in Fig. 6, deletion of V5-,
V11-, and V12-bearing thymocytes proceeds in the absence of both
B7-1 and B7-2. A small, but statistically insignificant, increase in
the percentage of CD4+ SP thymocytes expressing
V5, V11, and V12 is observed in the B7 DKO mice, but the
levels of these cells are significantly lower than those observed in
the absence of CD40 or CD40L. Thus, the failure to observe a
substantial effect on SAg-mediated thymic deletion in the absence of
either CD28 or B7 indicates that CD40-CD40L interactions do not rely
solely on signals derived from B7-CD28 interactions to affect negative
selection.
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The fate of potentially self-reactive thymocytes that escape SAg
-mediated deletion in the thymus of CD40L KO mice was explored by
determining frequencies of V5-, V11-, and V12-expressing T
cells in the spleens of CB6F1 CD40L KO mice.
Interestingly, despite the failure to observe negative selection in
CD4+ SP thymocytes, peripheral
CD4+ T cells expressing self-reactive Vs were
deleted in CD40L KO mice (Fig. 7). This
was not the case for peripheral CD8+ T cells
where deletion did not occur and where percentages of V5, V11,
and V12 remain elevated in CD40L KO mice (Fig. 7). Thus,
CD8+ T cells that normally would be deleted in
mice expressing endogenous SAgs were present in the periphery of CD40L
mice, whereas CD4+ T cells expressing these same
Vs were deleted. Analysis of CD40 KO mice yielded similar results
(data not shown). These results suggest that the deletion of
SAg-reactive cells is occurring in CD4+ cells
subsequent to down-modulation of CD8, either late in thymic SP
development or in the periphery. To distinguish between these two
possibilities, CD4+ SP thymocytes from
CB6F1 CD40L KO mice were stained with HSA to
differentiate mature (HSAlow) from immature
(HSAhigh) SP cells (14). Fig. 8 shows that the frequencies of
CD4+ SP thymocytes expressing V5, V11, and
V12 are significantly decreased as cells progress from the
HSAhigh to HSAlow stage in
the thymus and are further decreased in the periphery.
HSAlow CD4+ SP thymocytes
comprise a minor proportion of total CD4+ SP
thymocytes; thus, deletion in this population is not apparent without
gating on HSAhigh and
HSAlow subsets. The frequency of
V10-expressing cells, which are not subject to SAg-mediated
deletion, is similar at all maturational stages. These data indicate
that a pathway exists for CD40-CD40L-independent negative selection of
mature SAg-reactive CD4+ T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The identity of the CD40- and CD40L-expressing cells which participate in the negative selection process is not yet well defined. Expression of CD40 has been demonstrated on thymic cortical epithelial cells as well as medullary epithelial and dendritic cells (15, 16, 17). However, it has been reported that CD40L expression appears to be restricted to relatively mature thymocytes localized to the medulla (15), suggesting that CD40-CD40L interactions participating in negative selection would have their effect at this stage of development. As discussed below, this is consistent with the observation that SAg-mediated deletion in CD40-CD40L-intact mice occurs in relatively mature thymocytes transiting from the double-positive to the SP stage (18, 19).
Among the known effects of CD40 signaling on APCs is the induction of increased expression of B7-1 and B7-2 molecules, the two known ligands for CD28. Foy et al. (3), in their original study of deficient negative selection in CD40L KO mice, found that thymic expression of B7-2 was substantially reduced in CD40L KO compared with WT mice. These authors raised the possibility that the role of CD40 signaling during negative selection might be to increase expression of B7 molecules, leading to increased interaction of B7 with CD28 and/or CTLA-4 on developing thymocytes. However, consistent with previous reports (20, 21), we have found that SAg-mediated negative selection proceeds normally in CD28 KO mice. Recently, CTLA-4 has been implicated in negative selection (22) and evidence to suggest the existence of other, yet undefined, B7 receptors has been obtained in studies of CD28-CTLA-4 DKO (23, 24). These findings suggested that, although negative selection is intact in CD28 KO mice, defects in negative selection might be observed in the absence of both B7-1 and B7-2. We have found, however, that SAg-mediated negative selection is not significantly affected in B7 DKO mice, demonstrating that B7-1 and B7-2 are not essential for negative selection and that the role of CD40-CD40L interactions during negative selection cannot be explained solely by increased interactions between B7 molecules and their ligands.
Previous studies in WT mice have indicated that negative selection of thymocytes specific for SAgs occurs during the double-positive to SP transition at a stage where the cells undergoing selection express both CD4 and CD8 (18, 19). This observation has been invoked to provide an explanation for the deletions observed in both CD4+ SP and CD8+ SP lineages in response to SAg that appear to be recognized by CD4-dependent MHC II-restricted T cells. Consistent with previous reports, we have demonstrated that the defect in negative selection observed in the absence of CD40 or CD40L results in dramatic increases in the numbers of self-reactive thymocytes maturing to both the CD4+ and CD8+ SP lineages (3, 4, 5). Interestingly, a late-acting deletional mechanism appears to prevent substantial accumulation of CD4+ cells in the periphery. The late deletion which occurs in CD40 and CD40L KO mice is not apparent until the transition from HSAhigh to a more mature HSAlow phenotype. It affects CD4+ but not CD8+ SP cells, plausibly because CD8+ T cells, lacking expression of CD4, are not susceptible to deletion in response to SAgs presented in the context of MHC class II molecules. In preliminary experiments, we find that this late CD40L-independent deletion can occur in the absence of CD28 or of both B7-1 and B7-2 molecules, as assessed in mice in which both the CD40-CD40L and CD28-B7 pathways have been genetically disrupted (data not shown).
It has been postulated that differences in the avidity of interactions between TCR and MHC plus Ag complexes could alter the stage of thymic differentiation during which negative selection occurs, with stronger interactions promoting earlier deletion (25). CD40-CD40L interactions could impact signals received by the TCR by affecting expression of MHC plus Ag complexes. Stimulation of CD40 on APCs results in enhanced presentation of MHC-II-Ag complexes due to increases in both MHC II levels and enhanced peptide loading of MHC II molecules (13, 26, 27). Thus, the absence of CD40 signaling could result in decreased presentation of superantigen as well as peptide-derived Ag and a subsequent delay in deletion of self-reactive T cells. CD40-CD40L interactions have previously been shown to be critical to deletion of TCR-transgenic thymocytes developing in the presence of cognate Ag (3). This indicates that the effects, if any, of CD40-CD40L interactions on Ag presentation within the thymus are not restricted to the SAg system.
Several recent reports have provided compelling evidence for the existence of multiple, redundant costimulatory pathways which may operate to promote deletion of SP thymocytes. Kishimoto and Sprent (2) reported that in vitro stimulation of immature (HSAhigh) CD4+ SP thymocytes with anti-TCR Ab plus mAbs specific for several potential costimulatory molecules (CD5, CD43, or CD28) resulted in increased cell death compared with treatment with anti-TCR alone. Thus, it would be expected that modulation of in vivo negative selection resulting in increased numbers of autoreactive, HSAlow mature thymocytes would require inhibition of several costimulatory pathways. This expectation is supported in recent work by Page (28), who found that accumulation of autoreactive, HSAlow, mature thymocytes was indeed apparent only when multiple costimulatory pathways were inhibited concurrently using blocking Abs directed to CD5, B7 and TNFR. The results of our studies presented here extend these previous findings by showing that deletion of SP thymocytes can proceed in the absence of CD40-CD40L interactions, indicating that the role of these costimulatory molecules, if any, is also redundant at this late deletional stage.
Thus, we conclude that negative selection during thymic development can proceed by at least two apparently distinct pathways that differ both in the stage in which deletion is manifest and in the requirement for CD40-CD40L interactions. In the first, deletion occurs before the SP stage and is dependent on CD40-CD40L interactions but does not require cell autonomous expression of CD40L. The second occurs at a later stage of differentiation as thymocytes transition from the HSAhigh to HSAlow maturational stage and does not require CD40-CD40L interactions.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: CD40L, CD40 ligand; SP, single-positive; KO, knockout; DKO, double-KO; WT, wild type; HSA, heat-stable Ag; SAg, superantigen; Mtv, mammary tumor virus.
Received for publication November 8, 2001. Accepted for publication January 14, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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6+ cells and down-modulation of either CD4 or CD8 on phenotypically distinct CD4+8+ subsets of thymocytes expressing intermediate or high levels of T cell receptor. Int. Immunol. 3:265.This article has been cited by other articles:
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J. E. Buhlmann, S. K. Elkin, and A. H. Sharpe A Role for the B7-1/B7-2:CD28/CTLA-4 Pathway During Negative Selection J. Immunol., June 1, 2003; 170(11): 5421 - 5428. [Abstract] [Full Text] [PDF]
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