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,§
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Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121;
Department of Biology, University of California at San Diego, La Jolla, CA 92093;
Department of Microbiology and Immunology and
§
Division of Digestive Diseases, Department of Medicine, University of California, Los Angeles, CA 90095; and
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Department of Surgical Pathology, Cedars-Sinai Medical Center, Los Angeles, CA 90048
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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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Currently, several mouse models facilitate the study of IBD. One of
these models involves the adoptive transfer of purified
CD4+CD45RBhigh T cells from
the spleen or lymph node into syngeneic or MHC-matched immune-deficient
recipients. After homing to the intestine, the donor-derived cells
express phenotypic markers characteristic of normal intestinal
lymphocytes (13), but within several weeks of the
transfer, the recipients show chronic inflammation of the intestine,
with greater severity in the colon. The
CD4+CD45RBhigh T cells have
been shown to preferentially produce proinflammatory cytokines, such as
IFN-
and TNF-
, following transfer, and those cytokines are
important for pathogenesis (6). Studies in this model and
others have shown that induction of colitis requires the presence of
enteric bacteria (13, 14, 15). Furthermore, in a related
model, T cells from mice prone to spontaneous colitis induce disease
following in vitro activation by bacterial Ags and transfer into
immune-deficient mice (16). Taken together, these
data suggest that cells activated by microbial Ags may play a role in
colitis induced by T cell transfer into SCID mice.
Analysis of the TCR repertoire of the immunodeficient recipients in the CD4+CD45RBhigh T cell transfer model provides an opportunity to characterize the T cell population present in the intestine of diseased individuals in a manner not possible in clinical studies on human patients. The advantages of this model include 1) the predictability of disease onset, 2) the use of inbred donors and recipients, and 3) a lack of resident mucosal lymphocytes in the recipients, so that all lymphocytes found in the intestine must have been recently derived from the donor population. Therefore, for this study we have used a variety of techniques, including the use of genetically modified RAG2-deficient recipients, the analysis of donor T cells in recipients at early time points following cell transfer, and, most particularly, the analysis of TCR diversity, to assess the distribution of Ag-reactive T cells following transfer, and their potential role in colitis pathogenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Donor (C57BL/6 x BALB/c)F1 (CB6F1) mice were purchased from Simonsen (Gilroy, CA) and The Jackson Laboratory (Bar Harbor, ME) and maintained either at the University of California-Los Angeles vivarium or the La Jolla Institute for Allergy and Immunology. Donors were used between 6 and 12 wk of age. C.B-17 SCID mice were bred in ventilated cage racks from stock originally obtained from the University of California-Los Angeles SCID mouse core facility, or purchased from The Jackson Laboratory and maintained at the La Jolla Institute for Allergy and Immunology vivarium. Recipients received adoptive transfers between 7 and 12 wk of age. Donors and recipients were always of the same sex. All mice were housed under specific pathogen-free conditions. MHC class II-/- mice and RAG2-/- mice were purchased from The Jackson Laboratory and were intercrossed and maintained for the duration of the study at the vivarium at the University of California-Los Angeles. Offspring were analyzed by flow cytometry using tail blood stained with FITC-conjugated anti-I-Ab clone 25-9-17 and PE-conjugated anti-CD3 clone 145-2C11 (PharMingen, La Jolla, CA). Class II-/-RAG2-/-mice were identified as those mice that failed to stain positively for both I-Ab and CD3.
Transfer of CD4+CD45RBhigh T cells
Spleens were removed from donor CB6F1 mice and teased into single-cell suspensions in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% FBS (HyClone, Logan, UT). Lymphocytes were isolated by separation of total cells on a lympholyte gradient (Cedarlane, Hornby, Canada), and enriched for CD4+ cells by running over a negative selecting column following the manufacturer’s protocol (Cytovax Biotechnologies, Edmonton, Canada). These CD4-enriched cells were stained with FITC-conjugated anti-CD4 clone RM4-4 and PE-conjugated anti-CD45RB clone 16A (PharMingen). The cells were sorted on a FACStar (Becton Dickinson, San Jose, CA) at either the University of California-Los Angeles Flow Cytometry Core Facility (Jonsson Comprehensive Cancer Center) or at the La Jolla Institute for Allergy and Immunology.
Groups of recipients were each injected i.p. with 4–5 x 105 sorted donor lymphocytes, obtained from a pool of six donor mice, in 100–200 µl of sterile PBS. One aliquot of sorted donor lymphocytes (4 x 105) was pelleted, snap-frozen in liquid nitrogen, and stored at -70°C until used for TCR repertoire analysis of the starting population. The recipient mice were weighed initially, then weekly thereafter. They were observed for clinical signs of illness, including hunched over appearance, piloerection of the coat, and diarrhea. Diseased animals were sacrificed for analysis between 5 and 7 wk posttransfer.
Isolation of intraepithelial and lamina propria lymphocytes
Mucosal lymphocytes were isolated according to a previously published method (13). Briefly, small and large intestines were removed and carefully cleaned from their mesentery, opened longitudinally, and flushed of fecal content. Intestines were then cut into 0.5-cm pieces, transferred into 250-ml Erlenmeyer flasks, and shaken three times at 200 rpm for 30 min each time at 37°C in HBSS without Ca2+ or Mg2+ and containing 1 mM DTT (Sigma, St. Louis, MO). The cell suspensions were passed through a 60-µm nylon mesh, and cells were pelleted by centrifugation at 1200 rpm. The cell pellets were resuspended in 20% Percoll (Pharmacia, Piscataway, NJ), layered over a discontinuous 40/70% Percoll gradient, and centrifuged at 900 x g for 25 min. Cells from the 40/70% interface were collected, washed, and resuspended in complete RPMI 1640 medium supplemented with 5% FBS. To isolate lamina propria lymphocytes (LPL), the remaining intestinal tissue was minced, transferred to 250-ml Erlenmeyer flasks, and shaken for 60 min at 37°C in complete RPMI supplemented with 5% FBS containing dispase at 1.5 mg/ml (Sigma). The cell suspensions were collected and passed through nylon mesh, and cells were pelleted by centrifugation at 1200 rpm. The cells were passed over a Percoll gradient and processed as described above for the preparation of intraepithelial lymphocytes (IEL). The cell suspensions were pelleted for a final time for both IEL and LPL, were snap-frozen in liquid nitrogen, and were stored at -70°C until mRNA extraction was performed. None of the fragment length analysis data shown was derived from analysis of a cell population smaller than 1.5 x 105 cells.
Analysis of intestine tissue sections
Tissue samples of 
5 mm were taken from the intestine and
fixed in 10% formalin. Fixed tissue was embedded in paraffin; 3-µm
sections were prepared and then stained with hematoxylin-eosin. Samples
were coded and scored by a pathologist blinded to the conditions under
which the experiment was conducted. A previously described scoring
system was used for the tissue sections (13). This system
incorporates five parameters, including the degree of inflammatory
infiltrate in the lamina propria, mucin depletion, epithelial
hyperplasia/atypia, number of IEL in epithelial crypts, and number of
inflammatory foci per 10 high powered fields. The maximum score is 14;
higher scores indicate greater pathology.
Flow cytometric analysis of lymphocytes
IEL and LPL were resuspended in PBS staining buffer containing
2% BSA and 0.02% NaN3. After preincubation for
15 min at 4°C with the blocking 2.4G2 anti-FcR mAb, the cells
were stained at 4°C for 30 min with labeled mAb. Samples were then
washed three times in PBS staining buffer. Tricolor-conjugated
streptavidin was added as secondary staining reagent for the
biotinylated mAb followed by two washes in staining buffer. The samples
were immediately analyzed at this point, or they were fixed in PBS
containing 1% paraformaldehyde and 0.02% NaN3
and stored at 4°C. mAbs used in this study include CyChrome- or
FITC-labeled anti-TCRß clone H57-597, FITC- or PE-labeled
anti-Kb clone AF6–88.5, FITC- or PE-labeled
anti-CD44 clone IM7, biotinylated anti-CD69 clone H1.2F3,
FITC-labeled or biotinylated anti-CD4 clone RM4-4, PE-labeled
anti-CD45RB clone 16A (PharMingen), and FITC-labeled anti-CD62L
clone MEL-14 (Caltag, Burlingame, CA). Flow cytometric analysis was
performed on a Becton Dickinson FACScan 440 flow cytometer at the La
Jolla Institute for Allergy and Immunology.
mRNA extraction and cDNA synthesis
mRNA was prepared using the QuickPrep micro mRNA purification kit (Pharmacia). The full quantity of mRNA obtained was used for single-strand cDNA synthesis. The mRNA was denatured for 10 min at 70°C, then incubated with (dT)15 (5 mM), dNTPs (1 mM each), RNasin (40 U; Promega, Madison, WI), and AMV reverse transcriptase (2 U; Roche, Mannheim, Germany) in the supplier’s buffer at 43°C for 1 h, followed by incubation at 53°C for 10 min.
PCR amplifications, primer extensions, and data analysis
PCR amplification was conducted in 50 µl using 1/30 to 1/40 of the cDNA with 2 U of Taq polymerase (Perkin-Elmer, Foster City, CA) in the supplier’s buffer. Sense oligonucleotides specific for each of the 23 Vß genes, and antisense oligonucleotides for Cß have been described previously (17). Forty cycles of PCR were conducted in a 9600 Perkin-Elmer Automate, with each cycle consisting of 94°C for 45 s, 60°C for 45 s, and 72°C for 45 s. Each PCR product was then used as a template for extension, or run-off, reactions with oligonucleotides labeled with fluorescent tag. Fluorescent primers used in this study include an internal Cß primer and primers specific for each of the 12 Jßs (17). The fluorescent run-off products generated varied in size depending on CDR3 length. Run-off products were subjected to capillary electrophoresis in an automated DNA sequencer (Applied Biosystems, Foster City, CA), and CDR3 size distribution and signal intensities were then analyzed with GeneScan software (Perkin-Elmer). The patterns observed contained up to eight size peaks, each spaced by three nucleotides, corresponding to in-frame transcripts. The area under each peak was proportional to the quantity of TCR transcripts of the corresponding CDR3 length in the sample.
Cloning and sequencing of select Vß-Jß rearrangements
Amplified PCR products were diluted 1/100 with H20, and 1 µl used as a template for amplification of selected Vß-Jß rearrangements. PCR was performed with the reagents and quantities described above, using a sense oligonucleotide specific for the Vß-chain of interest and an antisense oligonucleotide specific for the Jß-chain of interest. Twenty-five cycles, each at 94°C for 45 s, 60°C for 45 s, and 72°C for 45 s, were completed in a 9600 Perkin-Elmer Automate. PCR products were analyzed on a 2% agarose gel stained with ethidium bromide to monitor the quality and quantity of the reaction products. Each Vß-Jß amplified product was then shotgun cloned with the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Resulting colonies were randomly selected, and cultures were grown. Plasmid DNA was isolated from bacteria cultures using Wizard Plus Miniprep kits (Promega). Sequencing reactions were performed with ABI Prism dRhodamine Terminator Cycle Sequencing Ready reaction kit (Perkin-Elmer) and analyzed on an automated sequencer.
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Results |
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Consistent with previous studies, RAG-/-
recipients of
CD4+CD45RBhigh T lymphocyte
transfers developed significant weight loss by 6 wk (Fig. 1
). Between 4 and 5 wk, the
RAG-/- recipients showed overt signs of
disease, including a hunched over appearance, piloerection of their
coat, and loose stool. We reasoned that if pathogenesis depended on
Ag-reactive T cells, elimination of the major Ag-presenting molecules
for CD4+ T cells, MHC class II, would abrogate
disease. To investigate this possibility, we generated class
II-/- RAG-/- mice and
transferred CD4+CD45RBhigh
T cells into these animals in parallel with identical transfers into
class II+RAG-/-
recipients. CD4+CD45RBlow T
cells from the same donor pool, which are known not to induce colitis
(18), were concurrently transferred into both class
II-/- RAG-/- mice and
class II+RAG-/- mice as
controls. By 8 wk all RAG-/- recipients of
CD4+CD45RBhigh T cells had
developed colitis and were sacrificed for analysis. By contrast, class
II-/-RAG-/-
recipients of
CD4+CD45RBhigh T cells
remained healthy and continued to gain weight, even at 18 wk
posttransfer, as did both sets of recipients of the
CD4+CD45RBlow T cell
population. Consistent with the lack of weight loss, there was no
evidence for inflammation of the intestinal tissue following the
transfer of CD4+CD45RBhigh
T cells to MHC class II-/- immune-deficient
mice. The average histologic score for the tissue sections of the class
II-/-RAG-/- recipients
was 4.7 ± 0.6, similar to the 4.0 score obtained when
disease-free, class II+ recipients of
CD4+CD45RBlow T cells were
examined. This is in contrast to an average score of 12.2 for class
II+ recipients of
CD4+CD45RBhigh T cells
(13).
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We analyzed the TCR repertoire in the large intestine IEL (LIEL) of seven diseased SCID recipients that had received CD4+CD45RBhigh T cells 5–7 wk earlier. This was performed initially by fragment length analysis, which gives an overview of the repertoire by indicating the size spectrum of the CDR3 regions present as well as the relative representation of the different CDR3 lengths. Gaussian, or bell-shape, distributions show a size spectrum of CDR3 regions that differ by up to 8 aa in length, with the mean lengths being most highly represented. Such Gaussian distributions of CDR3 lengths are typical of polyclonal populations. Previous observations have demonstrated that situations characterized by the expansion of a specific clone(s) or the presence of an oligoclonal population have always been revealed with fragment length analysis as a perturbation in the Gaussian CDR3 size profile (17, 19, 20, 21). The sensitivity of fragment length analysis allows for easy identification of clonal expansions, capable of detecting expansions found in frequencies as low as two members per 104 cells over a polyclonal background (22).
Analysis of a sorted
CD4+CD45RBhigh donor
population before transfer showed that its TCR repertoire has the
typical Gaussian profile. mRNA extraction and cDNA synthesis were
performed on an aliquot of
CD4+CD45RBhigh cells saved
from pooled donor cells that were transferred into recipient mice E, F,
and G. Twenty-three Vß-specific Cß PCR reactions were conducted,
and the product of each PCR was visualized by primer extension
reactions using an internal fluorescently labeled Cß oligonucleotide.
The CDR3 size profile for each Vß was Gaussian, having five to seven
peaks, each spaced by three nucleotides, corresponding to in-frame
transcripts (Fig. 2A). More
detailed analysis of the Vß repertoire can be achieved by analyzing
the CDR3 length distribution with Jß gene usage taken into
consideration. This is achieved by performing 12 separate primer
extension reactions on the initial Vß-Cß PCR product, with each
reaction using a fluorescently labeled Jß oligonucleotide specific
for a different Jß gene (17). Analysis of the donor
population in this manner further confirmed its polyclonality, as the
CDR3 size distribution for each Jß primer extension reaction was also
Gaussian (data not shown).
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A shows representative CDR3
profiles for five Vßs from LIEL of a single SCID recipient (mouse B).
There is evidence for predominant single or double peaks for Vß3,
Vß11, and Vß18, while Vß15 shows a Gaussian profile. The CDR3
size distributions for this and one other Gaussian Vß profile were
analyzed for each of the 12 Vß-Jß rearrangements to determine
whether the populations in question were truly polyclonal. In both
cases analyzed, the profiles generated using the fluorescently labeled
Jß primers were not Gaussian. An example of the results of the Jß
extension reactions is shown in Fig. 2B for the Vß15
repertoire. The normal preferential bias in the recombination machinery
to use Jß2 family members more often than Jß1 members
(23) was not found in this sample. For this Vß15
repertoire, Jß1 rearrangements are favored over Jß2, and Jß1.5
and Jß2.4 rearrangements were not detected. Numerous primer extension
reactions with different IEL populations demonstrated such preferential
reduced Jß usage, although the Jß genes that were not detected
varied from one instance to the next. Furthermore, the generation of
predominant CDR3 lengths other than the typical 8-aa mean by different
Vß-Jß rearrangements also argued against weak signal strength
producing the observed result. Therefore, all data obtained are
consistent with an oligoclonal TCR repertoire in the IEL of diseased
SCID recipients. Recipients of the same CD4+CD45RBhigh cells do not share common, expanded T cell clones
Two different donor pools of
CD4+CD45RBhigh T cells were
used. Each pool was formed by combining spleen cells from six donor
mice. Mice A, B, C, and D received cells from one donor pool, and mice
E, F, and G received T cells from a second pool generated separately.
Fig. 3A shows the complete set
of CDR3 profiles for Vß-Cß amplifications from mouse E. As noted
above, perturbations in the CDR3 profile were commonly found in
recipient mice and were categorized as either expansions or
pseudo-Gaussian profiles. We define expansions as those profiles with
no more than two predominant peaks. If there is a single predominant
peak, to be classified as an expansion it must comprise at least 50%
of the area under the profile curve, and if there are two predominant
peaks, they must contain at least 70% of the area. We classified
profiles as pseudo-Gaussian distributions if they were characterized by
a perturbation in the CDR3ß profile, i.e., one or more peaks that
demonstrate either more or less representation of a given CDR3 than
would be expected from a typical Gaussian profile. These
classifications were used in the comparison of the Vß repertoires of
the LIEL of seven recipient SCID mice, which are compiled in Fig. 3B. The data indicate that Gaussian profiles were obtained
in only a minority of the cases in each recipient. The number of true
Gaussian profiles, indicative of polyclonality, is likely to be much
less because, as noted above, distributions that appear Gaussian by
Vß-Cß amplification tend not to be Gaussian following Vß-Jß
amplification.
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, and they involve Vß1, Vß4, Vß9,
Vß11, Vß14, Vß16, and two predominant rearrangements for Vß2,
one that is 7 and one that is 8 aa in length. Interestingly, with the
exception of the Vß4 and Vß11 rearrangements, these common lengths
were found both in some mice that received donor cells from pool 1
(mice A–D) and some recipients from pool 2 (mice E—G; see Fig. 4).
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) that there is some sharing of the predominant
Jß used for particular Vß genes when different mice are compared,
but this was not true in all the mice that had a predominant fragment
of the same size following Vß-Cß amplification. Only three
Vß-Jß combinations were present in at least three recipient mice.
These include, with the CDR3 lengths shown in parentheses,
Vß2-Jß1.4 (7 aa), Vß2-Jß2.1 (8 aa), and Vß11-Jß2.1 (9 aa).
Both rearrangements involving Vß2 were selected for further study by
nucleic acid sequence analysis on DNA amplified with specific Vß and
Jß primers and cloned without size selection. The Vß14-Jß2.5
(9-aa) rearrangement and the Vß9-Jß2.7 (9-aa) rearrangement, each
present in only two recipient SCID mice, were also analyzed in this
way. Surprisingly, analysis of >50 sequences revealed only one shared
sequence between any two recipient mice, involving a Vß2-Jß2.1
rearrangement. In mouse F, five of nine of the Vß2-Jß2.1
rearrangements sequenced had a CDR3 of 8 aa that coded for the protein
sequence TGGNYAEQ. This identical nucleic acid and protein sequence was
also found in mouse B, in one of a total of 5 Vß2-Jß2.1 sequences
that were performed. All five sequences in mouse B had CDR3 lengths of
8 aa. However, a Vß2-Jß2.1 sequence found in 3 of 5 cases in mouse
B was not identified in the nine sequences in mouse F. Furthermore, in
mouse A, none of the 10 Vß2-Jß2.1 sequences studied matched those
sequences identified in either mouse B or mouse F. In summary, the analysis of both the Vß-Cß and Vß-Jß extension reactions provided only a limited number of cases of clones potentially shared between recipients, and the nucleic acid sequence data corroborated these findings and indicated that the majority of expanded Vß-Jß rearrangements with the same CDR3 length in different recipients are not identical. Therefore, while we cannot formally exclude the possibility that there are a significant number of clones common to different SCID recipients, the data indicate that the highly expanded clones in one recipient are not likely to be highly expanded in other recipients.
Lymphocytes in different sites of an individual mouse share similar TCR repertoires
We analyzed the TCR repertoire of donor-derived cells found in
different sites to determine whether there is a unique TCR repertoire
in the large intestine, which is the primary site of inflammation.
CDR3ß profiles from mice A, B, D, and G were generated from IEL and
LPL in the small and large intestines and from T cells in the spleen.
In total, 28 analyses permitted comparison of the large and small
intestinal compartments with the spleen, and an additional 31 analyses
allowed comparison between at least two intestinal compartments, with
both the LIEL and small intestine IEL (SIEL) represented in each case.
Representative data illustrating the CDR3 length profiles for three
Vß genes are shown in Fig. 5.
Comparison of the TCRß repertoire of the lymphocytes found in the
large intestine with those of other intestinal compartments and the
spleen reveal some similarities in the CDR3 distribution patterns. For
Vß18, a dramatic expansion of a TCR clone with a CDR3 length of 6 aa
in LIEL is likewise present in large intestine lamina propria (LLPL),
SIEL, small intestine lamina propria (SLPL), and spleen. Occasionally
observed, however, is a less striking TCR clonal expansion in LIEL, as
shown in Fig. 5 for Vß4 and Vß8.3. In the majority of the examples
in which we could compare the large and small intestinal compartments,
the CDR3 profiles were virtually identical. Exceptions to this tended
to exhibit greater CDR3 length diversity in the large intestine than
the small intestine.
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shows a compilation of
these CDR3ß protein sequences and the frequency with which they were
detected in various compartments, and Fig. 6 presents a detailed analysis of Vß15
rearrangements from a single mouse. Strong clonal expansions, as
detected by a predominant fragment length in different locations of the
same recipient, in fact gave identical sequences. For example, the
6-aa-long Vß18-Jß1.1 rearrangement that was predominant in all
compartments tested from mouse B (middle row, Fig. 5)
corresponded to only one nucleotide sequence (13 of 13) in all five
compartments (Table I). Based upon these data, we conclude that a
single clone was predominant among the Vß18 rearrangements found
throughout this mouse. Similar examples of such widely distributed and
prevalent clones include the Vß8.1 and Vß8.3 rearrangements from
mouse A and the Vß4 rearrangements from mouse D (Table I).
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). There is a clear clonal expansion of a 10-aa-long CDR3 in
SIEL and SLPL (Fig. 6, left). While a 10-aa CDR3 length is
readily detected in the spleen, LIEL, and LLPL, it is less predominant
due to the presence of two or three other CDR3 size peaks (Fig. 6).
Using run-off reactions with Jß-specific primers, the rearrangement
of Vß15 to Jß1.1 was determined to be the major rearrangement
responsible for the 10-aa CDR3 TCRß clonal expansion (data not
shown). DNA sequences obtained from 37 clones resulting from
amplification with Vß15- and Jß1.1-specific primers, but not
selected for the 10-aa size, also are shown in Fig. 6. The sequence
data are arranged in rows according to increasing CDR3 length and also
in columns according to the distribution of the different sequences in
this SCID recipient. Fourteen different sequences were obtained, and
three patterns for the distribution of the sequences emerged, including
sequences that were found to be shared between the spleen and intestine
(middle column, three different sequences in 12 total
clones), sequences found in various parts of the intestine only but not
in the spleen (right column, three examples), or sequences
unique to either the spleen (left column, four total)
or unique to one part of the intestine (left column,
four total). In this case, sequences shared between the large intestine
and spleen were not found in the small intestine, but this pattern was
not typical (Table I).
The expanded clone with a 10-aa CDR3 had the sequence RETGGPNTEV. Not
surprisingly, it was abundant in the small intestine (4 of 6
sequences), but while it was not found in the spleen (0 of 16
sequences) it also could be found in the large intestine (3 of 15). Two
other Vß15 CDR3 sequences that were detected only in the intestinal
compartments corresponded to the 7- and 8-aa CDR3ß lengths predicted
by fragment length analysis. Other sequences, listed in the
middle column of Fig. 6, were shared only between the spleen
and the LIEL or LLPL. It should be noted that 10-aa-long CDR3 regions
with protein sequences different from those of the predominant clone in
the small intestine were found, including one sequence found in spleen,
LIEL, and LLPL and one found in spleen only. These data demonstrate
that clonal identity, even in different locations in an individual,
cannot be strictly inferred from shared CDR3 lengths. Additional data
in Table I show the sequence and prevalence of expanded clones in
different sites. The degree of diversity may be somewhat higher
overall in the LIEL and spleen, but the data illustrate the
dissemination of prevalent clones throughout both the large and small
intestines and the spleen.
Related, but nonidentical, Vß sequences suggest an Ag-driven T cell expansion
The relatively high degree of similarity among the Vß15-Jß1.1
sequences provides some evidence for Ag-driven selection of particular
T cell clones. The common features include a CDR3 length of 10 aa, and
a 4-aa motif in the N-D-N region, which includes arginine followed by
an acidic amino acid, followed by a polar amino acid and then a
glycine. Additionally, the sequence RDRGRNTEV, found only in the
spleen, differs from another sequence found in spleen and elsewhere
only by lack of the tryptophan at the beginning of CDR3. Together,
these common length and sequence features are found in 15 of the 37
(41%) Vß15-Jß1.1 rearrangements (Table II).
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, which is
a compilation of sequences identified for the indicated Vß-Jß
rearrangements in different mice. Reiterative sequences found in four
or more sites in an individual recipient are indicated by a b
superscript and also can be found in Table I. Strikingly, many of the
N-D-N region protein sequences are very similar for a given Vß-Jß
rearrangement in a single mouse. For example, the Vß8.1-Jß1.2
rearrangement in mouse A demonstrated a 92% detection frequency of one
clone with a CDR3 protein sequence SRTANSDY. The only other CDR3
identified for this rearrangement differs by one amino acid, and it was
found in 1 of 13 total sequences identified. Likewise, the
Vß8.3-Jß2.7 rearrangements in mouse A have a 72% frequency of the
CDR3 protein sequence SDDDWVSYEQ. Two other 10-aa sequences were
identified, HDDDWVSYEQ and SVDDWVSYEQ, both with a DDWV protein
sequence in the N-D-N region. Together these constitute 84% of the
total sequences found. As described previously, a more subtle pattern
was identified for Vß15-Jß1.1 in mouse B. The shared CDR3 motifs
found in one recipient were not found in the other recipients. We
therefore consider it unlikely that the shared motifs that we observed
are due to a bias in the recombination machinery, which should be
revealed in all recipients. Rather, they probably reflect differences
in antigenic selection in individuals. CD4+CD45RBhigh T cells appear activated in both spleen and intestine early after transfer
The data obtained from both fragment length measurements and
nucleotide sequence analysis indicate that there is a significant
degree of sharing of the TCR repertoire of expanded clones between
various compartments of the intestine and the spleen of a diseased
recipient. This suggests that activated or memory T cells should be
found in both the spleen and the intestine. We therefore used flow
cytometry to phenotype donor-derived T cells in SCID recipients at
relatively early time points following cell transfer. Suspensions of
spleen cells and IEL were analyzed 7 or 11 days following transfer of
CD4+CD45RBhigh T cells from
CB6F1 mice (H-2d x
H-2b) into C.B-17 (H-2d)
SCID mice. This F1 into immune-deficient parental
strain transfer system permits the marking of donor-derived
(H-2b) T cells that can be distinguished from the
leaky production of T cells by the SCID host. In this transfer there is
neither a graft-vs-host nor a host-vs-graft response. Colitis generally
does not become severe until around 40 days after transfer.
Donor-derived T cells (Kb+
TCRß+) were detected by multicolor flow
cytometry, and expression of the activation markers CD69, CD62L, and
CD44 was investigated in this gated population. Representative data
from a single recipient mouse of three tested at 11 days posttransfer
are presented in Fig. 7. For comparison,
analysis of CD4+CD45RBhigh
T cells from two naive CB6F1 spleens was
performed in parallel. Based on expression of high levels of CD44,
expression of CD69 by the majority of the T cells, and the lack of
CD62L, the donor-derived (Kb+) lymphocytes appear
to be activated in both spleen and LIEL by day 11. The percentage of
gated cells that are CD44+ shifts dramatically
from 10.1% in a representative naive CB6F1
spleen to 98.2 and 96.5% of donor-derived cells in LIEL and spleen,
respectively, of the SCID recipient. A similar shift is observed in the
percentage of CD69+ cells in the gated
populations of the naive CB6F1 spleen compared
with the analysis 11 days posttransfer. Likewise, the percentage of
gated cells that are CD62L+ decreased from 87.6%
in the naive spleen to 0.4% in LIEL and 4.4% in spleen. The data from
mice at 7 days after transfer indicate that donor lymphocytes are
already activated at this time, but the extremely low numbers of
donor-derived cells that can be recovered at this time point made it
difficult to acquire enough Kb+
TCRß+ cells for a statistically reliable
analysis. We therefore conclude that at the earliest time points at
which significant T cell numbers can be isolated, activated T cells can
be found in both the intestine and the spleen.
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Discussion |
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Previously, we showed that T cells in the intestine of the diseased SCID mice have an activated phenotype, and it was shown that a normal bacterial flora is required for the efficient establishment of T cells in the host intestine (13, 26). It remained possible, however, that bacteria have a nonspecific activating effect on the transferred T cells. Here we show that expression of MHC class II molecules by the host is required for colitis pathogenesis, consistent with a role for TCR-mediated recognition in this process. Furthermore, we used an extremely sensitive technique, fragment length analysis, to achieve the first detailed description of the TCR repertoire in the intestine of mice with colitis. We find that the TCR ß-chain repertoire in IEL and LPL of SCID mice with CD4+ CD45RBhigh-induced colitis is characterized by the presence of predominant or expanded clones. If a superantigen were involved, by contrast, we would expect a predominant Vß with diverse CDR3 lengths. We did not find evidence for a predominant Vß following PCR amplification, however, and in an earlier publication flow cytometric analysis did not reveal the presence of a predominant Vß in the intestinal T cells of diseased SCID recipients (27). Similarly, comparative TCR repertoire studies on twins discordant for Crohn’s disease argued against the participation of superantigens in IBD in humans (28). Nucleic acid sequence analysis of T cells in the immune-deficient recipients demonstrates the presence of rearrangements that share amino acid motifs within the N-D-N region of the CDR3 of the ß-chain, providing further evidence for the Ag-driven selection or expansion of particular T cell clones. The presence of related clones with similar ß-chain N-D-N sequence motifs in an individual argues strongly against a random or homeostatic proliferation of lymphocytes in the "empty" SCID mouse as the explanation for a shared repertoire in different sites. Although these data taken together provide cogent evidence for an Ag driving the population of the host intestine with T cells, it remains possible that some of the T lymphocytes present in the mucosa are bystander cells.
Comparison of the TCR repertoires between recipients of cells from the same donor pool revealed a surprising paucity of shared or public clones in the intestine. Although this finding does not provide support for the hypothesis that there is a single predominant Ag in colitis pathogenesis, it does not exclude this possibility. It has been found that there is a relatively low level of sharing of the TCR repertoire even in unimmunized inbred mice (29), which might be due to stochastic elements in the Ag receptor gene rearrangement and selection processes. In addition to differences in the naive repertoire, recent studies demonstrate that small differences in the time of encounter with Ag are critical to the preferential expansion of particular T cell clones (30). Therefore, it remains possible that exposure to similar sets of Ags can generate different responses, even in genetically identical individuals.
Analyses of human IBD patients have provided some evidence for shared clones in peripheral blood CD4+ lymphocytes of identical twins concordant for Crohn’s disease (31) and for shared sequence motifs in activated T cells and CD8+ T cells of the intestinal mucosa from unrelated individuals with IBD (32, 33). Similar to the results from our analysis of diseased mice, however, in all these cases private or individual TCR expansions greatly outnumber the public clones identified.
An oligoclonal TCR repertoire has been reported in both normal mouse and human IEL and LPL (34, 35, 36). Although a few reports do not agree with this finding (27, 37), in these cases a detailed study of CDR3 sequences was not conducted. Interestingly, the limited diversity of the TCR repertoire in the intestine of inbred mice with colitis and the striking differences between recipient mice precisely parallel reports on the TCRß repertoire of IEL from normal mice (36, 38). These similarities suggest that the process of establishing a T cell population in the SCID intestine may in part reflect processes that form the repertoire of IEL and LPL in normal mice.
Predominant clones in the large intestine of an individual tend to be found in other regions of the body as well. The CDR3 length profiles of the LIEL showed a great deal of similarity to those in other regions of the intestine, including LPL, as well as the spleen, in any given animal studied. Sequence analysis confirmed the presence of systemic clonal expansions, and flow cytometric data demonstrate that CD4+ T cells in the intestine and the spleen tend to have an activated phenotype, even at relatively early time points following cell transfer. These data are consistent with either a systemic activation of T cell clones or a rapid and systemic distribution of T cells that have been activated in a particular site. Dendritic cells in the intestinal mucosa have been shown to be capable of migrating to sites outside the intestine (39, 40), providing a possible mechanism for the systemic presentation of intestinal Ags that might be relevant for colitis pathogenesis, such as those from bacteria. Alternatively, if there were a single site for the initial activation of the transferred T cells, for several reasons that site is unlikely to be either the epithelium or lamina propria of the intestine. First, there are data indicating that transferred T cells require antigenic stimulation to migrate to the intestine (41). Second, work in mice with joined circulatory systems, or parabiotic mice, demonstrates that IEL mix poorly between the parabiotic partners, although LPL recirculate to some extent (42, 43). Therefore, a putative site of initial activation would probably be in some organized lymphoid tissue. As Peyer’s patches were difficult to visualize in the recipients, this site is perhaps the mesenteric lymph nodes or the spleen.
The systemic distribution of predominant clones in the recipient mice stands in contrast to the localization of disease to the large intestine. The large intestine may become inflamed because of the higher bacterial load or because local factors in this environment might be more permissive of the Th1 responses that drive colitis. Additionally, we cannot formally exclude the possibility that subtle differences in the TCR repertoire in the large intestine might be critical for pathogenesis. These differences for the most part do not encompass the highly expanded clones, and therefore, we consider this possibility unlikely. It is more likely that the T cells in the large intestine are slightly more diverse than those in the small intestine simply because it is the site of disease. Once disease is initiated by activated clones, other T cells could be recruited into the large intestine nonspecifically by the production of cytokines and other inflammatory mediators. Additional experiments will be required to determine where the pathogenic T cells are initially activated in the CD4+ CD45RBhigh transfer model and why inflammation is concentrated in the large intestine.
It is not known whether the systemic distribution of activated T cells in the CD4+ CD45RBhigh transfer model also is characteristic of pathogenic T lymphocytes in human IBD. Expanded clones have been found among the CD4+ T cells in peripheral blood of patients with Crohn’s disease (31), however, and these clones persist for at least 1 yr. The relevance of the expanded clones in PBL for disease pathogenesis has not yet been established, but these data are consistent with the possibility that pathogenic cells in IBD are not localized exclusively in the intestine. Based upon these findings, we speculate that analysis of the diversity of the activated T cells in peripheral blood of IBD patients could shed insight into the diversity and specificity of pathogenic T cells.
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2 Address correspondence and reprint requests to Dr. Mitchell Kronenberg, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address:
3 Abbreviations used in this paper: IBD, inflammatory bowel disease; RAG, recombinase-activating gene; LPL, lamina propria lymphocytes; IEL, intraepithelial lymphocytes; LIEL, large intestine IEL; LLPL, large intestine LPL; SIEL, small intestine IEL; SLPL, small intestine LPL; CDR3, complementarity-determining region 3.
Received for publication October 12, 1999. Accepted for publication December 17, 1999.
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ß T cells in the colonic lamina propria of scid mice with colitis. Eur. J. Immunol. 26:1156.[Medline]
and the CD8 ß TCR-/ß murine intestinal intraepithelial T lymphocytes: evidence for the random emergence of T cells. J. Exp. Med. 180:1345./ß in the intestinal epithelium: evidence for distinct circulation patterns of gut- and thymus-derived T lymphocytes. J. Exp. Med. 176:187.This article has been cited by other articles:
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  M. W. Steinberg, O. Turovskaya, R. B. Shaikh, G. Kim, D. F. McCole, K. Pfeffer, K. M. Murphy, C. F. Ware, and M. Kronenberg A crucial role for HVEM and BTLA in preventing intestinal inflammation J. Exp. Med., June 9, 2008; 205(6): 1463 - 1476. [Abstract] [Full Text] [PDF]
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G. Srikrishna, O. Turovskaya, R. Shaikh, R. Newlin, D. Foell, S. Murch, M. Kronenberg, and H. H. Freeze Carboxylated Glycans Mediate Colitis through Activation of NF-{kappa}B J. Immunol., October 15, 2005; 175(8): 5412 - 5422. [Abstract] [Full Text] [PDF]
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J. S. Bryson, L. Zhang, S. W. Goes, C. D. Jennings, B. E. Caywood, S. L. Carlson, and A. M. Kaplan CD4+ T Cells Mediate Murine Syngeneic Graft-versus-Host Disease-Associated Colitis J. Immunol., January 1, 2004; 172(1): 679 - 687. [Abstract] [Full Text] [PDF]
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Z. Trobonjaca, F. Leithauser, P. Moller, H. Bluethmann, Y. Koezuka, H. R. MacDonald, and J. Reimann MHC-II-Independent CD4+ T Cells Induce Colitis in Immunodeficient RAG-/- Hosts J. Immunol., March 15, 2001; 166(6): 3804 - 3812. [Abstract] [Full Text] [PDF]
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A. V. MacDougall, P. Enders, G. Hatfield, D. C. Pauza, and E. Rakasz V{{gamma}}2 TCR Repertoire Overlap in Different Anatomical Compartments of Healthy, Unrelated Rhesus Macaques J. Immunol., February 15, 2001; 166(4): 2296 - 2302. [Abstract] [Full Text] [PDF]
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