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CUTTING EDGE |
Department of Biology and the Cancer Center, University of California at San Diego, La Jolla, CA 92093
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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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I reasoned that several costimulatory molecules might be cooperatively regulating negative selection, which would account for the conflicting data surrounding examination of any one of them. Here, the role of coreceptors in negative selection was examined by using a fetal thymocyte organ culture (FTOC) system. FTOC preserves the thymic environment via culture of a whole thymus with intact cortex, medulla, and endogenous APC, but one can still manipulate thymic selection by the addition of soluble mediators. In addition, I used models of negative selection, either with or without TCR transgenes, that do not involve an inflammatory response. This issue is absolutely crucial to the study of negative selection, as thymic deletion induced by the addition of Ag or anti-TCR Abs in the presence of mature T cells is complicated by the production of proinflammatory cytokines (9). I found that several costimulatory molecules that regulate mature T cell activation cooperatively participate in negative selection of the CD4+ T cell lineage. Blocking the contributions of these molecules prevented negative selection and induced the appearance of autoreactive thymocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AND TCR-transgenic (Tg) mice have been previously described (10). CD5- and CD28-null mice on the C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME).
Cell culture
Thymuses were removed from fetuses at day 16–17 of gestation
and cultured for 4–5 days as described (11). Following
culture, thymocytes were released by straining through nylon mesh.
Surface expression of CD4, CD8, TCR-V
11, and heat stable Ag (HSA)
was determined by Ab staining and flow cytometry, as described
(2, 11).
Abs and reagents
Anti-CD5 (53-7.3) was a generous gift from Dr. Gary Starling (Bristol-Meyers Squibb, New York, NY). Abs to CD80 (16-10A1), CD86 (GL1), and H-2Kb were purchased from PharMingen (San Diego, CA); rabbit anti-mouse TNF was purchased from Genzyme (Cambridge, MA). Anti-H-2Kb and hamster Ig (HIg; Jackson ImmunoResearch, West Grove, PA) were used at 60 µg/ml. The other Abs were titrated and used at concentrations that caused optimal maturation of CD4+ thymocytes in FTOC: anti-TNF sera at 1.25–2.5% and all others at 10–20 µg/ml.
Proliferation assays
Production of mature thymocytes capable of responding to Ag was
monitored by assessing thymocyte proliferation. A total of 1.25 x
105 thymocytes from FTOC were cultured for 3–4
days with 3 x 105 mitomycin C-treated
splenocytes from B10.A mice with or without the addition of Ag (the
88-103 COOH-terminal peptide of moth cytochrome c (MCC)).
For autoproliferative responses, 2.5 x 104
thymocytes were cultured for 5 days with 10 U/ml IL-2 and mitomycin
C-treated APC that were enriched for dendritic cells by collagenase
digestion of splenocytes and subsequent centrifugation over dense BSA
(12), as described (13). A dose-response of
103–105 APCs was used in
these cultures, but for purposes of brevity, only the response to the
highest APC dose is reported in Fig. 4
. The cells were pulsed with 1
µCi of [3H]methyl-thymidine (New England
Nuclear, Boston, MA) for the final 18 h of culture, and isotope
incorporation was determined.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the effect of coreceptors on thymocyte negative
selection, I first used the well-characterized AND TCR-Tg system. These
mice express a Vß3/V11-TCR that recognizes cytochrome c
peptides bound to H-2E class II MHC molecules (10).
Thymocytes bearing this TCR are positively selected on
H-2Ab (AND.b mice) such that a large population
of CD4+ cells develops in the thymus. Conversely,
if AND.b mice are crossed with B10.S(9R) mice (AND.b/9R), then the
presence of H-2As causes a dominant loss of
CD4+ thymocytes (14, 15). I used
FTOC of AND.b/9R thymuses to investigate the molecules that contribute
to negative selection. This system is advantageous because if one can
block negative selection to H-2As, then positive
selection on H-2Ab would result in the appearance
of mature CD4+ thymocytes. For example,
anti-CD40L treatment of AND.b/9R neonates (3) or FTOC
(data not shown) rescued the development of CD4+
thymocytes.
Previous reports indicated that CD5-null mice have altered thymic
maturation (16). Moreover, signals from CD28
(6) or TNFR (5) can cause thymocyte death in
vitro in conjunction with a TCR stimulus. Thus, I used Abs to CD5, TNF,
and the CD28 ligands B7-1 and B7-2 to block the interactions of these
molecules with their ligands. Although the identity of CD5L is
controversial (17, 18, 19), anti-CD5 blocks the
interaction of CD5L with CD5 (17). Anti-B7-1 and
anti-B7-2 likewise block T cell activation (20, 21),
and the anti-TNF sera blocks TNF cytolytic activity
(22). Fetal thymuses from AND.b/9R mice at day 17 of
gestation have very few CD4+ cells (data not
shown). After 4 days in FTOC, some CD4+ cells
developed (Fig. 1, top), similar to the
low level of development seen in AND.b/9R adults (14). A
population of CD8+ cells was also present, though
this population was never affected by the Ab treatments. However, when
these thymuses were cultured with Abs to CD5, B7-1 plus B7-2 (5 + B),
and TNF (5 + B + T), then a striking rescue of CD4 maturation occurred
(Fig. 1, top). Both the percentage and number of
CD4+ cells increased in these cultures, and the
CD4+ cells appeared to be mature as they
expressed low levels of the HSA (CD24) marker (Fig. 1, bottom) and high levels of the AND TCR (data not shown). Ag
reactivity should also correlate with CD4 maturation, and the
thymocytes from the cultures exhibiting rescue of CD4 maturation showed
increased proliferation to Ag (Fig. 1, middle). The blocking
Abs did not cause an increase in the
CD4+CD8+ (DP) population in
these cultures. This result was expected because negative selection in
neonatal AND.b/9R mice does not affect the DP population, but rather
occurs at the transition between the DP and CD4+
stages of development (14, 15).
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, bottom). Another possibility was that the blocking Abs were acting not by rescuing thymocytes from negative selection, but rather by stimulating proliferation of the small population of CD4+ cells that develop in the AND.b/9R thymuses. However, as expected from previous reports of the effects of anti-B7-1 and anti-B7-2 on T cell activation (20, 21), these Abs blocked AND thymocyte proliferation to Ag (data not shown). Moreover, the rescued CD4+ thymocytes were not proliferating, as measured by incorporation of bromodeoxyuridine (data not shown).
Finally, I tested the effect of these Abs on thymuses from mice
undergoing only positive selection (AND.b). Although AND.b mice
eventually develop a large population of CD4+
cells, the number of CD4+ cells in AND.b or
AND.b/9R thymuses is equivalent at birth (AND.b, 0.76 ±
0.39 x 106 CD4+;
AND.b/9R, 0.76 ± 0.15 x
106 CD4+; n
= 4–6 mice). The CD4+ population then declines
in AND.b/9R mice but increases in AND.b mice (14, 15).
Because the two types of mice have the same number of
CD4+ cells early in their development, AND.b FTOC
is an ideal control for any effects the blocking Abs may have that are
not due to rescue from negative selection. In other words, because the
CD4+ population is small in fetal/neonatal AND.b
mice, we could easily see an increase in this population if the
blocking Abs were somehow making positive selection more efficient.
However, the blocking Abs did not induce an increase in the
CD4+ population in AND.b mice (Fig. 1, bottom). These results strongly argue that the Abs to CD5,
B7-1, B7-2, and TNF cause an increase in the CD4+
population in AND.b/9R FTOC by specifically rescuing thymocytes from
negative selection on H-2As and subsequently
allowing them to be positively selected.
To determine the individual contributions of CD5, the B7 molecules, and
TNF to negative selection, fetal thymuses from AND.b/9R mice were
cultured with various combinations of the Abs to these molecules, and
the amount of CD4 maturational rescue was determined (Fig. 1, bottom). In general anti-CD5 alone (5) or anti-B7-1
plus anti-B7-2 alone (B) induced only a small rescue of the
CD4+ population; anti-TNF alone (T) rarely
induced CD4 rescue. However, the combination of 5 + B or 5 + T induced
CD4 maturational rescue in >90% of the cultures. Statistical analysis
(Student’s t test) of results from paired culture
conditions from 32 experiments with AND.b/9R thymuses confirmed these
observations: treatment of FTOC with 5 + B or 5 + T was significantly
different from treatment with 5, B, or T alone
(p < 0.05), and treatment with 5 + B + T was
most significant (p < 0.005). Interestingly,
the combination of B + T could not rescue CD4+
cells from negative selection (data not shown). Thus, it appears that
the block in negative selection is not due to simply an accumulation of
adhesive interactions, but that CD5 plays a central role that is
augmented by CD28 or TNFR.
Rescue from negative selection in other systems
I next investigated whether CD5, B7-1, B7-2, and TNF were involved
in other examples of negative selection. First, the effect of the
blocking Abs on negative selection induced by graded doses of a defined
Ag was examined. AND.b mice were crossed to B10.A mice (AND.b/a), which
express the H-2Ek molecule necessary for
presentation of the MCC peptide recognized by the AND TCR. Note that at
day 16 of gestation, there are no CD4+ thymocytes
in AND.b/a thymuses that could be stimulated to induce an inflammatory
response (data not shown). The development of
CD4+ thymocytes in FTOC is ablated in the
presence of Ag (Fig. 2, top,
HIg control). However, the blocking Abs rescued the
CD4+ cells from Ag-induced negative selection,
even at high doses of MCC. In this negative selection model, Ag also
induces DP cell death, and the blocking Abs rescued a significant
portion of the DP population from Ag-induced death as well. At 1000 nM
MCC, only 23% of the DP population survived, whereas 60% survived in
the presence of 1000 nM MCC plus 5 + B + T.
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, bottom). As in the AND.b/9R cultures, the rescued
CD4+ cells were not proliferating (data not
shown). Interestingly, Abs to CD5, B7-1, and B7-2 (and TNF, data not
shown) were not able to rescue CD8 maturation (Fig. 2, bottom). These results suggest that CD5, CD28, and TNF are
specifically involved in MHC class II-dependent negative selection.
Finally, I determined the effect of the blocking Abs on mice deficient
for either CD5 or CD28. Based on the results above, CD5-null mice
should show rescue from negative selection just by blocking interaction
of CD28 with B7-1 and B7-2. Correspondingly, CD28-null mice should show
rescue from negative selection just by blocking interaction of CD5 with
its ligand. This is exactly what was observed. Thymuses from
CD5+/- mice showed little or no rescue of CD4
maturation when cultured with anti-B7-1 and anti-B7-2 (Fig. 3, top). In contrast, the CD4
population in thymuses from CD5-/- mice treated
with anti-B7-1 and anti-B7-2 increased to levels obtained when
wild-type thymuses were treated with 5 + B. Analysis of data from seven
experiments confirmed that the difference in recovery of mature
HSAlowCD4+ cells between
CD5+/- and CD5-/- mice
treated with anti-B7-1 and anti-B7-2 was significant
(p < 0.03). Likewise, thymuses from
CD28+/- mice showed little rescue of CD4
maturation when cultured with anti-CD5. However, anti-CD5
treatment of CD28-/- thymuses caused CD4
maturational rescue equivalent to that obtained when wild-type thymuses
were treated with 5 + B. Again, the difference in recovery of mature
HSAlowCD4+ cells between
CD28+/- and CD28-/- mice
treated with anti-CD5 was significant (p <
0.01; five experiments). These results confirm that CD5 and CD28
cooperatively contribute to negative selection: when one receptor
is missing due to genetic ablation, blocking interaction of the other
receptor with its ligand(s) rescues thymocytes from negative selection
and allows them to mature into CD4+ cells.
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It has previously been shown that cells that escape negative
selection are autoreactive (23). Thus, as a final test of
maturational rescue, I examined whether the CD4+
cells from these cultures were able to respond to syngeneic APC in an
autoproliferation assay (13). Thymocytes obtained from
AND.b/9R FTOC cultured with the blocking Abs showed increased
proliferation in response to B10.S(9R) APC but not to C57BL/6 APC (Fig. 4, left). This result is
exactly what would be expected if AND thymocytes are being rescued from
negative selection on H-2As. Even though both
H-2Ab and H-2As are
expressed in AND.b/9R FTOC, the thymocytes are autoreactive only to APC
expressing the negatively selecting ligand,
H-2As. Thus, these results lend solid support to
the notion that the blocking Abs rescue cells that were destined for
negative selection. I next tested autoreactivity in the C57BL/6
cultures. As previously reported (13), significant
proliferation to autoAPC can be observed even in normal mice under
these conditions (HIg control; Fig. 4, right). However,
thymocytes obtained from C57BL/6 FTOC cultured with the blocking Abs
consistently exhibited increased proliferation in response to auto-APC
(Fig. 4, right). Taken together, these results show that
CD5, CD28, and TNF contribute to class II MHC-dependent negative
selection and that blocking the contributions of these molecules can
block negative selection and so induce the maturation of autoreactive
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These data are readily explained by the participation of multiple coreceptors in thymocyte death, and they are consistent with previous results showing that CD40L is involved in negative selection (3). Although the effect of CD40 stimulation on CD5L expression is not known (17, 19), B7-1, B7-2, and TNF are up-regulated by CD40-stimulation of APC (4) and B7-2 expression was reduced in CD40L-null mice (3). CD54 (ICAM-1) is also up-regulated on CD40-stimulated APC, and a recent report showed that CD54-null mice have a partial defect in negative selection (25). Negative selection due to class II MHC/peptide complexes is likely caused by the sum total of stimulation received from the TCR plus costimulatory molecules that are induced by CD40 stimulation of thymic dendritic cells. CD40 stimulation of peripheral dendritic cells is required to initiate an immune response (4). Thus, to avoid autoimmunity, the population of naive T cells needs only to be purged of cells that react with self-peptides on activated dendritic cells. By using CD40-activated coreceptors during both negative selection and the initiation of an immune response, the immune system has an excellent system in place for avoiding autoimmune responses.
There has been much debate as to what percentage of thymocytes that are
positively selected then undergo negative selection because the
affinity of their TCR for self-peptide/MHC complexes is too high.
Estimates of negative selection among the positively selected
CD4+ population have ranged from <5%
(23) to >50% (26, 27). We were initially
surprised by the large proportion of CD4+ T cells
rescued when normal C57BL/6 FTOC were treated with the blocking Abs
(Fig. 2). However, the 2- to 3-fold increase in the CD4 population in
these cultures correlates exactly with the 2- to 3-fold increase in
mature T cell production that is seen in mice that lack negatively
selecting APC in their thymuses (27). Thus, the results
presented here also imply that a high percentage of thymocytes that are
positively selected do not exit the thymus due to negative selection.
The observed increase in autoreactivity in these cultures supports this
notion (Fig. 4). These results are significant to the study of
autoimmunity, as the contribution of negative selection vs peripheral
tolerance to autoimmune diseases is not known. Interestingly, medullary
thymic epithelium expresses proteins that were originally thought to be
tissue specific or developmentally regulated (28).
Moreover, autoantigen expression in the thymus correlates with
resistance to some autoimmune diseases (29, 30, 31). Taken
together, these results suggest that negative selection is a
significant contributor among the mechanisms that the immune system has
developed to avoid autoimmunity. The identification of specific
coreceptors that regulate negative selection and autoreactivity
provides a new perspective for future explorations on the development
of autoimmune disease.
Note added in proof.
Kishimoto and Sprent have also demonstrated that several coreceptors regulate negative selection (J. Exp. Med. 190:65,1999).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dawne M. Page, Department of Biology and the Cancer Center, University of California at San Diego, La Jolla, CA 92093-0687. E-mail address:
3 Abbreviations used in this paper: FTOC, fetal thymic organ culture; Tg, transgenic; HIg, hamster Ig; MCC, moth cytochrome c peptide; DP, CD4+CD8+; HSA, heat stable Ag.
Received for publication June 1, 1999. Accepted for publication August 2, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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