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Department of Immunology, University of Washington, Seattle, WA 98195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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complex or pre-TCR (1, 2). This test of
in-frame ß-chain rearrangement sends the cells into a proliferative
cycle, results in the down-regulation of the lymphoid-specific
recombination machinery (recombination-activating gene
(RAG)3 1, RAG2, and TdT),
and renders the TCRß locus inaccessible, possibly by methylation or
nucleosome assembly. The outcome of this developmental program is to
redirect the attention of the recombinase complex to the TCR locus.
If the subsequent successful rearrangement of the TCR locus is
accompanied by an appropriate low affinity interaction of the TCR with
MHC molecules in the thymus, RAG1 and RAG2 expression are extinguished,
terminating further recombination (3), and the cells
become mature CD4 or CD8 single-positive cells (4). The
lag time between acquiring a functional TCR and processing the signals
that drive positive selection is such that allelic exclusion at the
TCR locus is more lax than at the TCRß locus.
Properly rearranged and selected TCR and ß transgenes effect
allelic exclusion of endogenous TCR genes by these same mechanisms
(5). Young C57BL/6 (B6) mice that carry in their germline
a productively rearranged Vß5 TCRß gene are characterized by high
transgene expression in both the CD4+ and
CD8+ intrathymic and peripheral T cell
compartments, indicating efficient allelic exclusion (6).
However, interaction with a peripheral tolerogen encoded by the
endogenous mammary tumor virus (Mtv)-8 drives the deletion of
transgene-positive CD4+ T cells, leading to an
inversion of the CD4:CD8 ratio in the lymphoid periphery (7, 8). This overall loss of CD4+ T cells is
accompanied by the appearance of
Vß5low-negativeCD4+ cells
expressing endogenous Vß chains on their surfaces (9),
cells referred to herein as Vßendo+. Although
the CD8+ T cell compartment remains uniformly
Vß5+, 40–50% of the peripheral
CD4+ T cells in older animals express alternate
TCR ß-chains, while maintaining transgene-specific mRNA expression
(C.J.M. and P.J.F., unpublished observations). In addition,
Vß5-Vßendo+CD4+
cells display a uniformly activated/memory phenotype (9).
The Vß5+CD4+ T cells
remaining in the periphery of older mice are anergic to stimulation by
plate-bound anti-Vß5 Abs, a fate not suffered by
Vßendo+CD4+ T cells,
which are readily stimulated by anti-Vß reagents (7, 9).
The mechanism by which a peripherally expressed tolerogen induces T cells to overcome allelic exclusion is suggested by our findings of RAG expression and recombination intermediates in Vß5low-negativeCD4+ T cells in Vß5 transgenic (Tg) mice (9). Thus, our data suggest that interaction of T cells with a tolerogen can trigger their entry into a tolerance pathway offering a choice: anergy and subsequent deletion; or TCR revision and subsequent reentry into the pool of functional peripheral T cells.
Our current understanding of this novel tolerance mechanism is limited by the lack of information on the frequency and origin of the T cells undergoing receptor revision. To explore these issues, we conducted a detailed molecular and cellular analysis of the TCR repertoire expressed by Vß5-CD4+ peripheral T cells in Vß5 Tg mice. Here, we show that the repertoire of the endogenous TCR ß-chains expressed in Vß5-CD4+ T cells from older Vß5 Tg animals is diverse both at the cell surface level and at the molecular level within a given family of V(D)Jß rearrangements. In fact, the endogenous TCRß repertoire in these "rescued" cells is nearly as diverse as the T cell repertoire in nontransgenic (nonTg) mice, although the stunted nontemplated (N) regions distinguish these sequences from those commonly generated in either the adult or fetal thymus. In addition, the appearance of Vßendo+CD4+ T cells is CD28 dependent. Taken together, these findings strongly suggest that tolerogen-driven TCR revision in Vß5 Tg mice occurs extrathymically. Its surprisingly high frequency demonstrates the remarkable flexibility of the immune repertoire.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Vß5+ Tg mice were generated on a B6 background (6) and are maintained as heterozygotes by crossing with B6 females purchased from The Jackson Laboratory (Bar Harbor, ME). Control mice were nonTg littermates. All mice were housed in a specific pathogen-free barrier vivarium at the University of Washington.
Cell preparation, Abs, and flow cytometry
Before staining, splenocytes were enriched for white blood cells by water lysis of RBCs, pooled with teased lymph node cells, and passed over nylon wool as previously described (9). Cells were stained with FITC-MR9.4 (anti-Vß5), CD4-PE, and a battery of biotinylated anti-Vß (Vß-bio) Abs followed by streptavidin Tri-Color (Caltag, San Francisco, CA). Live CD4+Vß5- cells were gated on the basis of forward and side scatter profiles and analyzed on a logarithmic scale for Tri-Color staining using a FACScan with CellQuest software (Becton Dickinson, Mountain View, CA). The markers for individual Vß Abs were set by the level of staining on the nonTg T cells. FITC-MR9.4, CD4-PE, MR12-5-bio (Vß13), MR10-2-bio (Vß9), and B20.6-bio (Vß2) were obtained from PharMingen (San Diego, CA). The remaining anti-Vß Abs were purified, biotinylated, and used as previously described (9).
PCR-RFLP
The basic protocol is as previously described (10).
The upstream primer for detecting rearrangements to the Vß8 family
(5'-TGGCAGCACTGAGAAAGGAGATAT) was generously provided by F. Livak.
This primer spans the EcoRV site in Vß8.1 and Vß8.2 and
changes one nucleotide in the sequence of Vß8.3 to create an
EcoRV site. The downstream primer anneals 3' of the Jß2
cluster (5'-CCTGGATCCAATTTGGGTGGGGAAGCGAG) and is end labeled with
[
-32P]ATP before the PCR. PCRs used 10 pmol
of the labeled Jß2 primer and 15 pmol of the unlabeled Vß8 primer.
Reactions of 30 cycles were purified on Qiaquick spin columns (Qiagen,
Valencia, CA), digested with EcoRV, separated by 6%
PAGE-urea, and sized using a sequencing ladder.
RT-PCR analysis
Vß5+CD4+ and Vß5-CD4+ populations were sorted to >90% purity using a FACStarPlus as described previously (9). Total RNA from sorted cells was reverse transcribed into cDNA with random hexamers using avian myeloblastosis virus reverse transcriptase (Life Technologies, Gaithersburg, MD). PCRs with hypoxanthine-guanine phosphoribosyltransferase (HPRT) primers (11) were performed on serial 3-fold dilutions of cDNA to determine equivalent amounts to use in subsequent PCRs with primers for murine TdT, RAG1, and RAG2. The TdT forward primer (5'-GAACAACTCGAAGAGCCTTCC) corresponds to exon 1, and the reverse primer (5'-CAAGGGCATCCGTGAATAGTTG) corresponds to exon 2 (12). The nested probe for the Southern blot was a 272-bp Xmn1-Bgl2 fragment from pDTS (12). The RAG primers and probes were previously described (9).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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A). As seen
previously (9), these CD4+ T cells
express uniformly high levels of TCR when screened with an
anti-TCRß Ab (H57-597, not shown). Multiple TCR ß-chains are
expressed within the
Vß5-CD4+ compartment
(Fig. 1B), and this level of diversity is representative of
each of the individual Tg mice screened (Fig. 1C).
Remarkably, the endogenous Vß repertoire of
Vß5-CD4+ T cells in
individual Vß5 Tg mice resembles the Vß repertoire expressed by
CD4+ T cells in nonTg B6 mice (Fig. 1C), with minimal mouse-to-mouse variation. As expected, the
CD8+ T cell compartment in these same mice does
not stain with any of these Vß Abs (data not shown). The diversity of
the endogenous Vß elements used by CD4+ T cells
in Vß5 Tg mice strongly suggests that the generation of
Vßendo+CD4+ cells is not
a rare event.
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A). Using primers specific
for the Vß8 gene family and a region immediately downstream of the
last Jß2 gene segment, we consistently found multiple bands in each
Tg sample analyzed (Fig. 2B). Interestingly, the size and
pattern of bands were unique for each Tg mouse analyzed and included a
subset of the bands seen in the nonTg samples. The PCR-RFLP products
seen in nonTg samples were similar from sample to sample and from
experiment to experiment. Thus, although the precise nature of the
endogenous TCRß rearrangements differs between nonTg and individual
Tg mice, these results indicate that even within a specific family of
Vß elements, there are multiple rearrangement events leading to the
expression of diverse endogenous TCRß genes in
CD4+ T cells undergoing TCR revision.
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C). The sequences, 50% of which were in-frame, were
characteristic of normal V(D)J junctions, including the loss of
nucleotides at the coding ends and the gain of both P and N
nucleotides. These results indicate that the junctional diversity of
the joined segments is maintained in Vß5 TCR Tg mice.
To assay directly for the expression of the enzyme responsible for N
nucleotide additions, we performed RT-PCR analysis with TdT-specific
primers. We found that peripheral populations of
Vß5low-negativeCD4+
cells, previously shown to express RAG1 and RAG2 (9), also
express mRNA specific for TdT (Fig. 3).
Titrations of cDNA indicated that TdT expression was lower in these
sorted cells than in thymocytes (not shown), analogous to the RAG
signal detected in the same populations (9). The lower
expression of these recombinase components could be due either to
diminished expression on a per cell basis or to a smaller number of
RAG+ cells in each population, possibilities that
these data cannot distinguish. These results demonstrate that the
peripheral CD4+ T cells capable of recombining
TCRß gene segments express all three of the developmentally
regulated, lymphoid-specific components of the V(D)J recombinase
complex (13), and more importantly, that each of these
components is functional and in contact with the TCRß locus
(9).
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). In contrast, endogenous Vß
sequences in Vß5 Tg mice contain only an average of 1.65 N
nucleotides (p < 0.04). Although these
sequences have shorter N regions than prototypical TCRß sequences
from adults, they are longer than those from fetal mice (Fig. 4), known
to lack TdT expression (15). Potential Vß-associated
differences in N region length cannot explain these results, given that
some of the published sequences are also of Vß8 origin (Fig. 4).
Thus, the endogenous Vß repertoire in individual Vß5 Tg mice is
indistinguishable from that expressed by nonTg B6 mice, but the TCRß
sequences are molecularly distinct from prototypical thymus-derived
sequences.
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A), the appearance of
Vßendo+CD4+ T cells is
restricted to wild-type mice (Fig. 5B). As expected,
CD8+ T cells expressing endogenous Vßs do not
accumulate in either CD28null or wild-type Vß5
Tg animals (Fig. 5B). These results, in combination with
data from B cell null mice (9), provide the most
compelling evidence for two distinct pathways taken by T cells
following recognition of the Mtv-8 tolerogen. These alternate pathways
are deletion, which requires neither B cells nor CD28 molecules, and
revision, which is both B cell and CD28 dependent.
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3:1 in aging Vß5
Tg mice, the periphery of which is characterized by a CD4:CD8 ratio
<1:5; 4) the percentage of mature CD4 single-positive cells expressing
Vß5 remains >95% in the thymus of aging Tg mice (7).
These observations, in combination with the data presented here,
strongly implicate the RAG-mediated TCR revision detected in peripheral
Vß5low-negCD4+ T cells as
a thymus-independent means for maintaining self tolerance and
generating a remarkably diverse TCR repertoire.
RAG reexpression in B cells has recently come under closer scrutiny
following the development of mice carrying a targeted replacement of
the RAG1 or RAG2 genes with a gene encoding green
fluorescent protein controlled by the endogenous RAG promotor
(17, 18, 19). These recent data suggest that peripheral
RAG+ B cells represent immature cells that have
failed to down-regulate RAG expression after leaving their
developmental compartment. Importantly, immature
RAG+ B cells can be found in the spleens of young
mice. These RAG+ B cells contain broken DNA
molecules characteristic of 
and 
chain gene rearrangements; have
an immature IgM, IgD, heat stable Ag, and B220 surface phenotype;
express the surrogate light chains 5 and
Vpre-B; and fail to express TdT
(20, 21, 22, 23, 24, 25). In contrast, the RAG+ T
cells in our system, although found in the spleen, have been isolated
only from mice >23 wk of age. These cells carry broken DNA molecules
characteristic of TCR ß-chain gene rearrangement, the T cell
equivalent of B cell heavy chain recombination. Importantly, thymocytes
that have divided while transiting from the double-negative to the
double-positive compartment would seal any breaks within the TCRß
locus. In addition,
Vßendo+CD4+ T cells
display a mature T cell surface phenotype (CD44+,
CD4+, CD8-,
CD62Llow, VLA4high), fail
to express the pT component of the pre-TCR (7, 9), and
express TdT (Fig. 3). These data suggest that the
CD4+ T cells in our system are not immature cells
that have failed to down-regulate RAG expression on positive selection.
Rather, these mature T cells are driven to reexpress RAG1, RAG2, and
TdT by encounter with a weak peripheral tolerogen, creating breaks
within the TCRß loci of mature cells that result in endogenous TCRß
expression, providing the cell an opportunity to avoid death.
One goal of our ongoing studies is to understand how the interaction
between a T cell and a single tolerogen can lead to either cell anergy
and death or cell rescue through TCR revision. Our current model is
that the T cell is nudged along one pathway or the other, based on the
strength or frequency of its interaction with tolerogen-expressing
cells. The diverse TCR -chain repertoire in Vß5-Tg mice
(8) provides the possibility of TCRs with differing
affinities for the tolerogen (26). A sufficiently weak and
chronic interaction of
Vß5+CD4+ T cells with the
peripheral tolerogen (perhaps on B cells) may cause down-regulation of
Vß5 surface expression and subsequent up-regulation of the
lymphoid-specific components of the V(D)J recombinase. We predict that
the cells continue to express RAG while they remain
TCR-. Once gaining a revised and functional TCR
ß-chain, we speculate that RAG expression would again be terminated.
This scenario is supported by recent observations in TCR and Ag
double-Tg mice, in which TCR internalization, RAG expression, and
rearrangement of endogenous TCR loci have been demonstrated in
thymocytes undergoing receptor editing (27). Additionally,
RAG expression and recombination intermediates have been seen in mature
human CD4+ T cells expressing low levels of TCR
(28). A direct test of this model in our system would
require quantitative analysis of single cells to assess the
relationship between TCRß and RAG expression levels.
Although our data do not directly address the anatomic site of TCR
revision, the following correlations suggest that
RAG+ T cells may be localized to germinal centers
(GCs), where they may undergo receptor revision followed by some type
of selection. First, CD4+ (but not
CD8+) T cells are known to enter GCs, and
CD4+ (but not CD8+) T cells
undergo TCR revision in Vß5 Tg mice (9). Second,
CD4+ T cells from neither B cell- nor
CD28-deficient Vß5 Tg mice express endogenous TCR ß-chains, despite
the fact that Vß5+CD4+ T
cells are efficiently deleted in these animals (Ref. 9 and
Fig. 5); GCs also fail to form in mice of these backgrounds
(29). Finally, GCs provide a selective environment for
Ag-reactive lymphocytes; any autoreactive T cells generated by TCR
revision are rendered anergic or deleted because
Vßendo+CD4+ T cells from
Vß5 Tg mice fail to proliferate on coculture with syngeneic B6
splenocytes (7, 9).
The nature of the signal(s) that might drive a cell to enter the GC microenvironment and undergo TCR revision and how this signal differs from those received by cells targeted for anergy or deletion are still unknown. Clearly, TCR revision is used frequently enough by peripheral, tolerogen-reactive T cells to create a remarkably diverse repertoire in the Vß5-CD4+ T cell compartment of aging Vß5 Tg animals. Our studies present important clues to the ongoing maintenance and diversification of the peripheral T cell repertoire.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Pamela J. Fink, University of Washington, Department of Immunology, Campus Box 357650, Seattle, WA 98195.
3 Abbreviations used in this paper: RAG, recombination activating gene; bio, biotinylated; B6, C57BL/6; GC, germinal center; HPRT, hypoxanthine-guanine phosphoribosyltransferase; Mtv, mammary tumor virus; N, nontemplated; nonTg, nontransgenic; Tg, transgenic.
Received for publication August 29, 2000. Accepted for publication September 26, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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ß T cell development in the thymus of normal and genetically altered mice. Curr. Opin. Immunol. 9:263.[Medline]
and ß T cell receptor alleles. Cell 69:529.[Medline]
-chain on T cell reactivity and tolerance to Mls-1 in T cell receptor ß-chain transgenic mice. J. Immunol. 151:556.[Abstract]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, Data from individual Tg mice on a wild-type B6
background; 
, data from nonTg B6 littermates. Data obtained from
Vß5 Tg CD28null B6 mice are denoted by a + symbol.
B, Transgene expression is gradually lost in
CD4+ peripheral T cells from wild-type but not from
CD28null Vß5 Tg mice. Cells from Tg mice collected and
stained as in A were gated for CD4+ or
CD8+ cells, and at least 2000 events were analyzed for
Vß5 expression. Bars represent data obtained from the indicated age
groups and averaged from individual Vß5 Tg mice on either a B6
wild-type or CD28null background. Sample numbers for each
group are as follows: n = 6–29 for 6- to 15-wk-old
mice; n = 10–29 for 16- to 25-wk-old mice; and
n = 18–37 for 26- to 35-wk-old mice. Error bars
represent the SD of the means.