Late Postnatal Expansion of Self-reactive CD8aa1 IntestinalIntraepithelial Lymphocytes in Mice*

BRADLEY S. PODDa, CAROLINE ABERGa, TIFFANY L. CHRISTOPHERa, FRANCISCO PEREZ-CANOb andVICTORIA CAMERINIb,†

aDepartment of Pediatrics, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA; bDepartment of Pediatrics and Center forImmunology, University of California, Irvine, Irvine, CA 92697, USA

(Received 30 November 2004; Accepted with revisions 2 December 2004)

The intestinal epithelium is unique in that it harbors auto-reactive T cells largely absent from the peripheralTCR repertoire in normal mice. Intestinal intraepithelial lymphocytes (IEL) expressing self-reactive TCRare mostly CD8aaþ cells in adult H-Y TCR RAG2/2 male mice homozygous for the restricting MHC Iallele, H-2D b. By contrast, in male mice heterozygous for the restricting and non-restricting MHC I allele,H-2D d (MHC F1, H-2Db/d), IEL are composed of CD8ab and CD8aaþ T cells. Here we demonstrate thatIEL in the immediate postnatal period of MHC homozygous male mice were mostly CD82 T cells, whileIEL in MHC F1 male mice were CD82 and CD8abþT cells. Regardless of the MHC I configuration and theability to support positive selection of CD8abþ cells in the thymus, the expansion of CD8aaþ IEL was alate postnatal event that followed a reduction in CD82 IEL. Furthermore, although in vivo treatment withthe specific peptide antigen resulted in an earlier accumulation of activated IEL, the expansion of CD8aaþ

IEL remained inefficient until late in postnatal life. Finally, as CD82 IEL stimulated with TCR agonistsin vitro, acquired expression of CD8aa, we propose that CD8aaþ IEL derive from CD82 IELintermediates. Whether CD82 IEL are CD8ab-lineage cells that escape deletion in the thymus or are T cellstargeted to the intestine from the thymus because of the early and high level TCR transgene expression inthis model, is not clear. The signals required for the expansion of CD8aaþ IEL are however, incomplete inthe immediate postnatal intestine. Determining the factors required for the expansion or retention ofCD8aaþ IEL bearing high affinity, self-specific TCR will further elucidate the in vivo role of these T cellsin intestinal homeostasis and perhaps, autoimmunity.

Keywords: Mucosa; T cells; MHC; Transgenic/ knockout mice; Cell differentiation

Abbreviations: Agpos APC, Smcy antigen positive APC isolated from male mice; DP, double positive;DN, double negative; H-Y TCR, TCR-transgenic mouse line reactive to H-Y Ag; IEL, intestinalintraepithelial lymphocytes; MHC F1, H-Y TCR H-2Db/b X H-2Dd/d; RAG, recombination activatinggene; SP, single positive; 7-AAD, 7-aminoactinomycin D

INTRODUCTION

Intraepithelial lymphocytes (IEL)3 in the small intestine of

mice are mostly CD8þ T cells that express CD8 as an ab

heterodimer or as an aa homodimer.[1 – 3] The CD8ab

heterodimer is expressed by TCR abþ IEL, while the

CD8aa homodimer is expressed by TCR abþ and TCR

gdþ IEL.[1,4,5] Despite different TCR isotypes, functional

similarities between CD8aaþ IEL suggest that this form

of CD8 defines or facilitates a common pathway in the

development or differentiation of IEL.[2,6] Although

comparisons between TCR abþ and TCR gdþ IEL are

limited, several differences in the development and

repertoire selection between CD8ab and CD8aaþ IEL

expressing the TCR ab are notable.[7 – 10] First, virtually

all CD8ab IEL and the majority of TCR abþ CD8aa

IEL, are absent in congenitally athymic nude mice.[1,11,12]

This suggests that precursors of these IEL are dependent

on epithelial cell-mediated selection in the thymus.

Precursors of CD8aaþ IEL may however, be selected

more efficiently in the fetal and early neonatal thymus,

whereas the development and selection of CD8abþ IEL

precursors may be less temporally restricted.[13,14] This is

interesting in light of the fact that most IEL in early

neonatal life are CD42, CD82 double negative (DN),

CD4þ CD82 and CD8abþ IEL whereas CD8aaþ IEL are

ISSN 0891-6934 print/ISSN 1607-842X online q 2004 Taylor & Francis Ltd

DOI: 10.1080/08916930400027094

*Supported by National Institutes of Health Grant AI44990.†Corresponding author. Tel.: þ949-824-1972. Fax: þ949-824-2305. E-mail: vcamerin@uci.edu

Autoimmunity, December 2004 Vol. 37 (8), pp. 537–547

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more abundant in later postnatal life.[15,16] Second,

CD8aaþ IEL persist in mice with targeted deletion of

the MHC class I genes K and D, while as expected,

CD8abþ T cells are markedly reduced[17 – 20] thereby

suggesting that the development and expansion of

CD8aaþ IEL do not require conventional MHC class I

molecules expressed in the thymus or periphery. Third, the

expression of self-reactive (forbidden) TCR Vb genes by

CD8aa IEL, but not CD8abþ IEL suggests that TCR with

high affinity for self-antigens may select CD8aaþ IEL or

their precursors in the thymus or intestine.[21 – 23]

Extensive studies in H-Y TCR transgenic mice also

support the notion that high affinity peptides positively

select precursors of CD8aaþ IEL.[24 – 27] The immediate

precursor of CD8aaþ IEL is however, still a matter of

debate. It has been hypothesized, that CD8aaþ IEL derive

from precursors that accumulate in the intestine after

escape from deletion in the thymus due to decrease or loss

in surface CD8ab expression (CD8ab2/low).[26,28] The

recent detection of CD8aaþ cells in the thymus of H-Y

TCR transgenic male mice and their development in

re-aggregate thymic organ cultures in the presence of the

agonist peptide, suggest that CD8aaþ IEL may be directly

downstream of the CD8aaþ cells selected in the

thymus.[29]

The unusual TCR specificity and unique developmental

kinetics of CD8aaþ IEL in mice with an unrestricted TCR

led to our evaluation of CD8aaþ IEL development during

postnatal life in the H-Y TCR transgenic mouse model.

In this study, we found that the expansion of CD8aaþ IEL

was a late postnatal event that followed the activation and

relative decrease in CD82 IEL. Moreover, CD82 IEL

stimulated with TCR agonists generated CD8aaþ T cells

during in vitro culture. Although CD8þ single positive (SP)

cells in the thymus of H-Y TCR MHC homozygous male

mice included cells that expressed CD8aaþ, CD8þ SP cells

in the thymus of MHC F1 male mice were always CD8abþ

cells suggesting that the generation of CD8aaþ IEL was

independent of this pathway in the thymus. Taken together,

our data suggest that CD8aa IEL derive from CD82 T cells.

Whether CD82T cells exit the thymus as a sole consequence

of early TCR a gene expression and efficient trafficking to

the intestine or are generated from CD8abþ cells that escape

deletion in the thymus or periphery needs to be determined.

Defects in this pathway may have implications for the

development of inflammatory bowel diseases and perhaps

play a role in the extra-intestinal manifestations of these

diseases in humans.

MATERIALS AND METHODS

Mice

Female and male B10.D2 RAG-22/2 (homozygous for the

non-restricting MHC I allele, H-2D d) and C57Bl/10

RAG 2 22/2 (homozygous for the restricting MHC

I allele, H-2D b) mouse strains homozygous for the H-Y

TCR a and b transgenes[30] (H-Y TCR, B10.D2 RAG-22/2

and H-Y TCR, C57Bl/10 RAG-22/2 , respectively) were

obtained from Taconic Farms (Tarrytown, NY).

The intercrosses of non-transgenic male or female

C57Bl/10 RAG-22/2 and B10.D2 RAG 2 22/2 mice

with H-Y TCR, B10.D2 RAG 2 22/2 or H-Y TCR,

C57Bl/10 RAG-22/2 mice, respectively, resulted in

progeny that were heterozygous for the restricting

(H-2D b) and non-restricting (H-2D d) MHC class I alleles

(MHC F1, H-2D b/d). Mice were housed in a laminar flow

barrier facility under specific pathogen free conditions. Mice

pregnant with MHC F1 or H-Y TCR, C57Bl/10 RAG-22/2

(MHC homozygous) progeny were identified and monitored

daily until delivery, which was assigned as day 0 of life.

Individual mice were examined at intervals beginning at two

weeks of age and continuing as noted in results. The

Institutional Animal Care and Use Committee approved all

animal protocols and procedures.

Preparation of Lymphocyte and Antigen Presenting

Cell Populations

Mucosal lymphocytes were prepared from the small

intestine of individual mice using our previously published

procedure.[7] Briefly, the small intestine was dissected from

its mesentery and washed in RPMI 1640 (Life Technol-

ogies, Inc., Grand Island, NY). The intestine was opened

longitudinally and the contents were removed before the

intestine was cut into 0.5 cm pieces. Mononuclear cells

were released from the epithelium by shaking in calcium

and magnesium free HBSS (Life Technologies, Inc.)

supplemented with 1 mM DTT (Sigma Chemicals,

St. Louis, MO) 3 times for 20 min at 250 rpm. Mononuclear

cells collected from the epithelial layer were filtered

through stainless steel mesh and enriched on a discontinu-

ous 20/40/70% Percoll gradient (Pharmacia Biotechnol-

ogy, Piscataway, NJ) by centrifugation at 900g for 20 min.

The resultant cell populations were washed in RPMI 1640

with 10% FCS and analyzed as described below.

Mononuclear cells were prepared from the thymus and

spleen following mechanical disruption of the tissue

capsules using a glass mortar and stainless steel mesh

screen (Sigma Chemicals). Red blood cells were depleted

from cell suspensions of the spleen by hypotonic lysis as

we have previously described.[31] The resultant mono-

nuclear cells from the spleen and thymus were suspended

in PBS mAb staining buffer containing 5% FCS, 0.02%

NaN3, (all from Sigma) with 5.0mg/106 cells of purified

CD16/CD32 mAb (BD Pharmingen, San Diego, CA).

Samples were stored at 48C until use as noted below.

Antigen presenting cells for use in vitro assays were

released from the spleen using the procedure noted above.

Resultant mononuclear cells were suspended in DMEM

complete media (10% FCS, 1% non-essential amino acids,

2 mM L-glutamine, 0.5% sodium pyruvate; all from Life

Technologies) supplemented with 10mg/ml gentamicin

(Sigma) and stored at 48C until use in co-culture assays

described below.

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Antibody Staining and Flow Cytometric Analysis of

Lymphocyte Populations

Mononuclear cells obtained from the thymus and

intestinal epithelium were suspended in PBS mAb

staining buffer at a concentration of approximately 1 £

106 cells=ml: Optimal concentrations of mAb directly

conjugated to FITC, PE, allophycocyanin or biotin were

added to each cell suspension in 100ml of staining

buffer and incubated at 48C for 30 min. After primary

antibody incubation, cell suspensions were washed in

PBS containing 0.02% NaN3 at 48C and suspended in

PBS mAb staining buffer with an optimal concentration

of avidin PE-Cy7 (Caltag Laboratories, San Francisco,

CA) or avidin PerCP (BD Biosciences, San Diego, CA)

to detect biotin-conjugated primary mAb. Cells were

incubated at 48C for 20 min and then washed in PBS

containing 0.02% NaN3 and fixed in PBS containing

0.2% NaN3 and 2% formaldehyde (both from Sigma)

prior to flow cytometric analysis. The mAb used for

these studies were: CD31 (145-2C11) PE or biotin; CD4

(YTS 19.11) PE; CD45 (30-F11) biotin; CD8a (53-6.7)

PE or APC and CD8b (53-5.8) FITC or PE and CD69

biotin (H1.2F3) (BD Biosciences and BioLegend,

San Diego, CA).

Mononuclear cell populations were analyzed on a

Becton-Dickinson FACS Calibur flow cytometer (BD

Immunocytometry Systems, San Diego, CA). Lympho-

cytes were gated from epithelial cells by characteristics

of size and granularity by the analysis of forward

scatter and side scatter properties. The FACS quadrants

were set from the analysis of cell populations stained

with single fluorescent mAb and isotype controls. The

data were displayed on two color dot plots or single

fluorescence histograms generated from the analysis of

cells electronically gated as noted in the Figure

Legends using the CellQuest computer program (BD

Immunocytometry Systems). Statistics for these data

were generated using the features of the software.

Compensation of fluorescence detectors was performed

on a weekly basis using single fluorescence-conjugated

beads and the FACS comp software (BD Immunocy-

tometry Systems).

Peptide, mAb and SEB Treatment of Mice

The Smcy-3 peptide KCSRNRQYL[32] was synthesized

on a Rainin Symphony Multiple Peptide Synthesizer.

Mice were injected i.p. with 100mmoles of Smcy-3

peptide daily in 50–100ml of PBS for a total of three

injections prior to harvesting cell populations at the

designated weekly intervals after birth. Purified CD31

(145-2C11) mAb (BD Biosciences) 2.5mg per 20 g body

weight and SEB 1.5mg/g of body weight (Toxin

Technologies, Sarasota FL) were injected in 100ml of

PBS daily by i.p. injection for two days before

harvesting IEL at four weeks of postnatal age as noted

in the results.

Sorting of T Cell Subsets and In Vitro Stimulation

Assays

CD82 T cells were isolated from the intestinal epithelium

and the spleen by FACS. Cell populations were prepared

for sorting by incubating with optimum concentrations of

CD31 PE, CD8b FITC and CD8a allophycocyanin for

30 min followed by washing in PBS with 10% FCS (Life

Technologies, Inc.). Prior to sorting, cells were suspended

in RPMI supplemented with 10% FCS. CD3þ T cells were

electronically gated and CD82 T cell subsets collected

with a half-log window between cell populations

expressing CD8a and CD8b on a FACS Vantage Cell

Sorter (BD Immunocytometry Systems). Sorted cells

(.99% purity upon reanalysis) were suspended at 1:0 £

106 cells=ml in complete DMEM and plated at 1:0 £

105 cells in half-area 96-well flat bottom culture dishes.

The wells were pre-coated with 10mg/ml of CD31 (BD

Biosciences) mAb suspended in PBS or seeded with APC

prepared from the spleen of male C57Bl/10 RAG2/2 mice

(Agpos APC). APC were included in a ratio of 10 to 1 of

either purified IEL or splenic T cells. Cell cultures were

incubated at 378C with 5% CO2 for 48 h. Following

culture, cells were harvested from wells and suspended in

PBS staining buffer with CD31 PE, CD8b FITC and

CD8a APC (all from BD Biosciences) as described above.

7-aminoactinomycin D (7-AAD) (Molecular Probes,

Eugene, OR) was added at final concentration of

10mg/ml following the manufacturers’ recommendations

for the exclusion of dead cells during flow cytometric

analysis. APC were excluded from analysis of T cells by

expression CD31 and by forward and side scatter profiles

by flow cytometry.

Statistical Analysis

The student’s two-tailed t-test was used to determine

whether the means between two samples were statistically

significant. p values less than or equal to 0.05 were

considered statistically significant.

RESULTS

The Earliest IEL are CD82 and CD8ab1T Cells,

whereas CD8aa1 T Cells Accumulate in LaterPostnatal Life

We examined IEL beginning at 2 weeks of postnatal age

when the intestinal epithelium can be separated from the

underlying lamina propria thereby allowing the reprodu-

cible isolation of IEL.[16] Although most IEL in H-Y TCR

adult male mice homozygous for the restricting MHC I

allele are CD8aaþ T cells,[24 – 26] this was not the case for

IEL in early postnatal life. In fact, at two weeks of

postnatal age, we found that more than 50% of IEL

isolated from H-Y TCR male mice lacked expression of

CD8a and CD8b (as well as CD4), and were therefore

CD42, CD82 DN cells whereas small, but nearly equal

THE ONTOGENY OF CD8þ IEL 539

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populations of IEL were either CD8aaþ or CD8abþ cells

(Fig. 1A). Similar proportions of CD8aaþ and CD8abþ

IEL were also found at 3 and 4 weeks of postnatal age,

whereas at 5 weeks of age, CD8aaþ IEL increased to 71%

of IEL while CD82 and CD8abþ IEL decreased.

Although a large proportion of IEL in MHC F1 male

mice were CD42, CD82 DN cells at 2 and at 3 weeks of

postnatal age, a greater proportion of IEL were CD8abþ

cells at all ages examined than when compared to MHC

homozygous male mice (Fig. 1B). For example, IEL at 2

weeks of postnatal age were composed of nearly equal

proportions of CD82 and CD8abþ T cells (49% versus

42% of IEL, respectively). Although CD8aa IEL were

present beginning at 2 weeks of postnatal age, they

represented less than 20% of IEL until 4 weeks of

postnatal age. After 4 weeks of age, CD8aaþ IEL

increased and by 5 weeks of age became nearly equally

abundant as CD8abþ IEL. CD8aaþ T cells never became

the majority of IEL in MHC F1 male mice as they did in H-

Y TCR male mice homozygous for the restricting MHC I

allele.

The differences in CD82 and CD8aaþ IEL, subsets

during the weeks after birth represented statistically

significant differences between 2 and 5 weeks of postnatal

age in both mouse strains (Fig. 1C and D). However,

unlike the reciprocal kinetics and the significant statistical

differences between CD82 and CD8aaþ IEL, CD8abþ

IEL did not vary significantly with advancing postnatal

age in either mouse strain. CD8abþ IEL were however,

always more abundant in MHC F1 male mice than in H-Y

TCR MHC homozygous male mice. No significant

differences in co-receptor expression were detected in

IEL isolated from mice at 5 weeks of postnatal age

compared to IEL at 6 weeks of postnatal age in either

MHC configuration.

CD31 IEL Expand during Postnatal Life in H-Y TCR

Mice with the most Significant Increase in CD81 IEL

Subsets

The differences in co-receptor expression by IEL during

the first weeks of postnatal life in H-Y TCR transgenic

male mice were also accompanied by an increase in the

number of IEL. We found that CD3þ IEL increased

significantly from 2 until 4 weeks of age of postnatal age,

after which time, further increases were not statistically

significant (Fig. 2A). When the number of IEL in each

co-receptor group were determined in MHC F1 male mice,

we found that CD8aaþ IEL increased more than 100 fold

between 2 and 4 weeks of age. In addition, although a

statistically significant increase in CD8abþ IEL occurred

between 2 and 4 weeks of postnatal age, the magnitude of

FIGURE 1 The phenotype of IEL in H-Y TCR male mice during the weeks after birth. IEL were isolated from individual MHC homozygous H-2Db/b

(A) and MHC F1 H-2Db/d (B) male mice H-Y TCR transgenic male mice at weekly intervals from 2 to 5 weeks of postnatal age. Multi-color flowcytometry was used to detect the expression of CD8a and CD8b by IEL electronically gated for CD3e and CD45 expression. The percentage of cells ineach quadrant is shown in the upper-right corner. Representative data from 2 to 8 individual experiments are shown. Co-receptor expression for IELisolated from MHC homozygous (C) and MHC F1 (D) male mice. IEL were prepared from individual mice and analyzed in separate experimentsincluding those shown in A and B. The mean percentage of CD3þ IEL expressing CD8aa (B), CD8ab (W) or lacking expression of CD8a, CD8b andCD4 expression, (CD82, O) are shown as a function of postnatal age at weekly intervals from two to six weeks. The error bars show the SD. *p , 0.05when CD8aaþ IEL at two weeks postnatal age were compared to CD8aaþ IEL at five weeks of age. †p ¼ NS when CD8abþ IEL at two weeks of agewere compared to CD8abþ IEL at five weeks of age. ‡p , 0.05 CD82 IEL at two weeks of age were compared to CD82 IEL at five weeks of age.No differences were found when IEL subsets at 5 weeks of age were compared to 6 weeks of postnatal age. The data were derived from 2 to 8experiments where mice were examined individually. The summary data include data shown in sections A and B above.

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this increase was less than for CD8aaþ IEL (Fig. 2B).

By contrast, although CD82 IEL were proportionally the

most abundant IEL subset in early postnatal life,

numerically this subset increased only 2 fold over the

period from 2 to 4 weeks of age. These results indicate that

significant CD8þ T cell accumulation occurs, either

through migration, differentiation, expansion or retention

during the early weeks of postnatal life. Moreover, co-

receptor expression by IEL varies significantly during this

period despite the restriction imposed by this monoclonal

TCR transgenic system.

Activation of IEL Precedes the Postnatal Expansion

of CD8aa1 IEL

We reasoned that the paucity of CD8aaþ IEL in early

postnatal life could result from an intrinsic defect in the

ability of a few CD8aaþ IEL or their precursors to expand

in vivo, perhaps because of a defect in the ability of the

environment to signal their development or expansion. To

explore this possibility, we pre-treated H-Y TCR mice

with Smcy-3 peptide 3 days prior to harvesting IEL during

weekly intervals after birth. This allowed us to examine

the effect of treatment on the development and

FIGURE 2 Comparison of the number of IEL in each co-receptor expression subset during postnatal maturation. (A). The absolute number of CD3þ

IEL as a function of increasing postnatal age from 2 to 6 weeks of postnatal age in MHC F1 male mice is shown. The number of IEL was calculated fromthe total mononuclear cell yield determined by light microscopy, corrected for the percentage of CD3þ cells determined by flow cytometry of gatedpopulations as described in Methods. All data were derived from individual experiments that analyzed individual mice. Each data point represents anindividual mouse in a minimum of two to a maximum of six experiments. *p , 0.05 when the number of IEL at two weeks of postnatal age wascompared to IEL at 3, 4, 5 and 6 weeks of postnatal age. †p ¼ NS when IEL at 3 weeks of age were compared to IEL at 4, 5 and 6 weeks of postnatal age.(B). The absolute number of CD8aa, CD8ab and CD82 IEL isolated from MHC F1 male mice is shown as a function of postnatal age from 2 to 6postnatal age in weeks. The number in each co-receptor subset was determined by correcting the total number of CD3þ IEL for expression of CD8a,CD8b or absence of either CD8 chain (CD82) as determined by flow cytometry. *p , 0:05 when CDaaþ IEL isolated at two weeks of postnatal agewere compared to CDaaþ IEL at any interval from 3 to 6 weeks of postnatal age. †p , 0:05 when compared to CD8abþ IEL isolated at greater than fourweeks of postnatal age. ‡p ¼ NS when CD82 IEL isolated at two weeks were compared to any other interval. The error bars show the SD. The data werederived from 2 to 8 experiments where mice were examined individually.

THE ONTOGENY OF CD8þ IEL 541

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differentiation of IEL subsets prior to reaching the

maximum number of CD8aaþ IEL noted at 5 weeks of

postnatal age in both mouse strains. We first examined

MHC F1 male mice, whose IEL were mostly CD82 and

CD8abþ T cells until 4 weeks of postnatal age. Although

CD8aaþ IEL increased after treatment with the cognate

peptide when compared to control mice, CD8aaþ IEL did

not increase to the maximal level observed at 5 weeks of

postnatal age (Figs. 3A and 1B). For example, at 2 weeks

of postnatal age CD8aaþ IEL represented 15% of CD3þ

IEL in peptide treated mice, nearly twice was found at this

interval in control mice. Whereas treatment at 3 weeks of

postnatal age did not result in an increase in CD8aaþ IEL

relative to control mice. Treatment with the cognate

peptide at 4 weeks of postnatal age was successful in

increasing CD8aaþ IEL to the levels normally found in

mice at 5 weeks of postnatal age. Treatment of MHC

homozygous male mice with peptide prior to 4 weeks of

postnatal age also resulted in small increases in CD8aaþ

IEL, whereas treatment with the cognate peptide at 4

weeks of postnatal age resulted in an increase in CD8aaþ

IEL to the level normally observed at 5 weeks of postnatal

age (Fig. 3C and data not shown). In the example shown,

treatment with the cognate peptide resulted in a nearly

3-fold increase in CD8aaþ IEL and a corresponding

decrease in CD82 IEL. Treatment of MHC homozygous

male mice with other TCR agonists resulted in similar

increase in CD8aaþ IEL and decrease in CD82 IEL.

We next compared the level of CD69 expression by IEL

during the weeks after birth and in response to treatment

FIGURE 3 TCR agonists activate IEL and promote the accumulation of CD8aaþ IEL in postnatal life. (A). H-Y TCR MHC F1 male mice wereinjected daily with 100mmoles of Smcy-3 peptide for three consecutive days prior to the harvest of IEL at 2, 3, 4, and 5 weeks postnatal age. IEL isolatedfrom individual mice and stained with mAb to CD3e, CD8a and CD8b that were examined as noted in Fig. 1. Representative examples of IEL preparedfrom individual mice treated at the weekly intervals noted are shown. (B). Aggregate data derived from the analysis of individual H-Y TCR MHC F1

male mice in peptide or control mice as shown in A, but were pooled for comparison. As in Fig. 1, multi-color flow cytometry was used to determine thepercentage of CD8aaþ, CD8abþ and CD82 IEL by electronically gating on CD3þ cells. Control mice injected with saline and are indicated by filledbars while open bars indicate peptide-treated mice. Error bars indicate SD. Results represent at least two experiments (weeks 2–4) except peptide-injected mice harvested at 5 weeks of postnatal age, where an individual mouse is shown. (C). MHC homozygous H-Y TCR transgenic male wereinjected with PBS (control), Smcy-3 peptide, CD3e mAb or SEB as noted in the Methods prior to the harvest of IEL at 4 weeks of postnatal age. IEL wereisolated from individual mice and then stained with mAb to CD3e, CD8a and CD8b. Two color dot plots of CD8a and CD8b expression by IELelectronically gated on CD3e T cells are shown. The examples are representative of at least 3 experiments examining mice individually. (D). Multi-colorflow cytometry was used to detect the expression of CD69 by all CD3eþ IEL isolated MHC F1 male IEL electronically gated for CD3e expression. CD69expression is shown in single-fluorescence histograms after electronically gating on CD3eþ IEL. The dashed line (– – –) indicates CD69 expression incontrol mice while the solid line (—) indicates CD69 expression by IEL isolated from peptide-treated mice. The percentage of positively staining IEL isshown in the respective populations in the upper left (control) and right hand (peptide treated) corner of each histogram. Representative data of at leasttwo experiments derived from the analysis of individual mice are shown. The peptide-treated group at 5 weeks is representative of an individual peptidetreated and control mouse.

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with the cognate Smcy-3 peptide. CD69 expression was,

on average lower on IEL at 2 weeks of postnatal age than

on IEL in later postnatal life (Fig. 3D and Table I).

In addition, we found that CD82 IEL expressed higher

levels of CD69 than did CD8abþ and CD8aaþ IEL. In all

cases, treatment with the exogenous peptide resulted in an

increase CD69 expression when compared to control

mice. Moreover, the increase in CD69 expression occurred

to a greater extent earlier in ontogeny than at later times

after birth when the level of CD69 had already increased

with normal maturation. Although treatment with

exogenous peptide resulted in an increase in CD69

expression by IEL subsets, the absolute number of IEL in

peptide treated mice was similar to the number of IEL

found in untreated and control mice (Fig. 2A and Table I).

These data demonstrate that the expansion of CD8aaþ

IEL is preceded by the accumulation of activated T cells in

the intestinal epithelium, the majority of which are CD82

IEL. Moreover, although treatment with the exogenous

peptide increased CD69 expression by all IEL subsets, the

degree of CD8aaþ IEL expansion appeared to be

constrained prior to 4 weeks of postnatal age and

paralleled the decrease in CD82 IEL. The increase in

CD8aaþ IEL was in both control and peptide treated mice

associated with an increase in CD69 expression,

suggesting that their differentiation is peptide (antigen)

driven.

CD82 IEL acquire CD8aa Expression whenCo-cultured with Antigen Positive APC and

after Cross-linking with CD31 MAb

The decrease in CD82 IEL (the majority of which were

CD69þ) and the increase CD69þ, CD8aaþ IEL following

treatment with TCR agonists in vivo, suggested that CD82

IEL were precursors of CD8aaþ IEL in situ. To test the

potential of CD82 IEL to give rise to CD8aaþ T cells,

purified CD82 IEL were co-cultured with APC isolated

from male, Smcy antigen positive, (Agpos APC) or were

stimulated with plate-bound CD31 mAb. CD82 IEL

stimulated with CD31 mAb and Agpos APC gave rise to

CD8aaþ T cells with few CD8abþ T cells. By contrast,

CD82 T cells from the spleen remained mostly CD82

although few CD8ab2/low cells were generated (Fig. 4).

These results demonstrate that freshly isolated CD82 IEL

contain T cells with an intrinsic ability to differentiate as

CD8aaþ T cells in response to agonist signals through the

TCR.

TABLE I Changes in the number of IEL and the expression of CD69 in MHC F1 male mice treated with the cognate peptide at weekly intervalsafter birth

# CD3þ IEL (106)* %CD69þ CD8aaþ IEL† %CD69þ CD8abþ IEL† %CD69þ CD82 IEL†

Postnatal Age (wks) Control Peptide Control Peptide Control Peptide Control Peptide

2 (n ¼ 3) 0.06 0.07 26 ^ 3 64 ^ 3 12 ^ 2 52 ^ 6 31 ^ 4 57 ^ 43 (n ¼ 3) 0.19 0.20 35 ^ 12 83 ^ 9 27 ^ 11 75 ^ 15 53 ^ 5 73 ^ 94 (n ¼ 2) 0.48 0.58 43 ^ 4 60 ^ 12 32 ^ 3 42 ^ 12 55 ^ 11 74 ^ 135 (n ¼ 1) 0.15 0.21 69 50 58 42 70 63

*Absolute number of CD3þ IEL £ 106 isolated per mouse was calculated from the total mononuclear cell yield determined by light microscopy, corrected for the percentageof CD3þ cells as determined by flow cytometry of gated populations stained with CD31, CD8b and CD8a mAb.†Percent of CD69 expression determined by flow cytometry for CD3þ cells electronically gated by co-receptor expression ^ the SDn ¼ number of experiments in which individual mice in control and peptide treated groups were compared

FIGURE 4 T cell differentiation assays following TCR stimulation in vitro. CD3eþ, CD82 IEL and CD82 T cells from the spleen were isolated to highpurity by FACS from H-Y TCR transgenic male mice. The post sort purity of a representative cell population after sorting is shown. CD82 T cell subsetswere incubated in triplicate wells with APC isolated from the spleen of RAG-22/2 , H-2D b male (Agpos APC) mice at a ratio of 10:1, or in wells pre-coated with plate bound CD3emAb (10mg/ml) for 48 h. The post-culture analysis of CD8 isoform expression is shown after gating CD31þ live cells by7-AAD exclusion. The percentage of cells staining positive in each dot plot is indicated. All data are representative of a minimum of two experimentsperformed in triplicate wells.

THE ONTOGENY OF CD8þ IEL 543

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Efficient Down-regulation of CD8b Expression in the

Thymus of H-Y TCR Mice is dependent on the Agonist

Self-peptide and two Copies of Restricting

MHC Class I Allele H-2Db

The majority of cells in the thymus of H-Y TCR

transgenic male mice homozygous for the restricting

MHC I allele after birth were CD42, CD82 DN cells

(70–76% of cells). The remaining cells included a small

population of CD4þ, CD8þ double positive (DP) (range

from 2–5%) and CD8þ SP cells (15–22%) (Fig. 5A).

Although most CD4þ, CD8þ DP and CD8þ SP cells at 1

week of age expressed nearly equal levels of the CD8a

and CD8b chain, progressive down-regulation of CD8b

expression occurred in excess of CD8a which gave rise

to CD8aþb2/low cells and CD8aaþ thymocytes.

CD8aaþ thymocytes were also evident in older mice.

Thymocytes in H-Y TCR MHC F1 male mice were also

mostly CD42, CD82 DN cells, although this subset

decreased with advancing postnatal age as CD4þ, CD8þ

DP (8–15%) and CD8þ SP (16–29%) subsets increased

(Fig. 5B). The percentage of CD4þ, CD8þ DP and CD8þ

SP thymocytes were always consistently higher in MHC

F1 male mice than in MHC homozygous male mice

indicating that negative selection and deletion of

CD8abþ lineage cells were less efficient in these mice

than in MHC homozygous male mice. Consistent with

this, CD8þ thymocytes in the CD4þ, CD8þ DP and

CD8þ SP subsets in MHC F1 male mice expressed higher

levels of CD8ab with few to no CD8aþb2/low and

CD8aaþ cells when compared to MHC homozygous

male mice. The maintenance of CD8ab expression was

also found in the thymus of female MHC homozygous

H-Y TCR mice. Taken together, these results demonstrate

a consistent and early postnatal role for the high affinity

self-peptide and two copies of the restricting MHC I

allele in promoting negative selection and deletion of

CD8abþ cells. The accumulation of CD8aaþ thymo-

cytes is unique to the thymus of MHC homozygous male

mice and therefore is likely not obligate for the

development of CD8aaþ IEL.

DISCUSSION

T cells expressing CD8aa were previously thought to

develop solely through extra-thymic pathways in the

intestine.[25,33] It is now clear that CD8aaþ T cells are

widely distributed in mice and humans and include T cells

that develop through conventional pathways in the

thymus.[31,34 – 37] Recent data demonstrate that CD8aaþ

cells bearing high affinity TCR in mice are selected in the

thymus in response to agonist peptides.[28,29,38] As similar

conditions generate CD8aaþ IEL in vivo, whether

CD8aaþ IEL are direct descendents of CD8aaþ

thymocytes or derive from a different cell intermediate

has not been determined.[29,39,40] To address this question,

we examined the kinetic relationship between IEL subsets

during postnatal maturation in H-Y TCR transgenic male

mice and in response to treatment with TCR agonists. This

enabled us to determine the kinetics of IEL colonization in

response to endogenous and manipulated TCR signals

during ontogeny. We found that CD8aaþ IEL achieved a

homeostatic maximum with similar kinetics during

postnatal maturation in both mouse strains regardless of

the MHC I configuration. In addition, CD82 IEL were

similarly abundant in early postnatal life of both mouse

strains and demonstrated an inverse kinetic relationship

with CD8aaþ IEL in vivo. The capacity of CD82 IEL to

generate CD8aaþ T cells in vitro following treatment

with TCR agonists supports the hypothesis that CD82 IEL

are precursors of CD8aaþ IEL. Thus, precursors of

CD8aaþ IEL may develop through pathways that are not

dependent on negative selection of CD8ab-lineage cells

in the thymus.

FIGURE 5 Analysis of CD8a and CD8b expression by thymocytesisolated from H-Y TCR transgenic mice. The thymus of MHChomozygous male (A), MHC F1 male and MHC homozygous female(B) mice were isolated at the postnatal ages indicated. After staining withfluorescence-conjugated mAb as noted in Methods, electronically gatedCD45þ thymocytes were analyzed for expression of CD4, CD8a, andCD8b. CD4 and CD8a expression by total thymocytes (upper panels inA and B) and the corresponding expression of CD8a and CD8b onCD4þCD8þ DP and CD8þ SP thymocyte subsets were determined byelectronic gating (lower panels in A and B). The percentage of cells inrelevant populations is shown adjacent to the box gate or in theappropriate quadrant. Data were derived from individual mice and arerepresentative of a minimum of two independent experiments.

B. S. PODD et al.544

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Several pathways must be considered for the origin of

CD8aaþ IEL. One possibility is that bone marrow

progenitors generate CD8aaþ IEL during development in

cryptopatch aggregates or in the epithelial lining of the

intestine, independently of the thymus. Although the

existence of this pathway in non-transgenic mice is still a

matter of debate,[41 – 43] a recent study suggests that

precursors of TCR abþ IEL (including CD42, CD82 DN,

CD4þ SP, CD8abþ SP and CD8aaþ SP subsets) must

traverse the CD4þ CD8þ DP point in the thymus.[37] The

severe restriction in the development of CD8aaþ IEL in

athymic H-Y TCR male mice supports the hypothesis that

precursors of CD8aaþ IEL originate in the thymus.[40]

Moreover, the development of CD8aaþ IEL in athymic

male RAG2/2 mice implanted with thymus fragments

from H-Y TCR male mice (our unpublished observations

and [28]) and their absence in athymic male RAG2/2 mice

transplanted with bone marrow from male H-Y TCR mice

(our unpublished observations) supports this conclusion.

A likely scenario in H-Y TCR mice therefore, is that

progenitors of CD8aaþ IEL derive from bone marrow

progenitors in the thymus. Whether cryptopatch aggre-

gates and the intestinal epithelium contribute in the post-

thymic differentiation of CD8aaþ IEL remains to be

determined.

The inverse kinetic relationship between CD82 IEL and

CD8aaþ IEL during postnatal maturation and the ability

of CD82 IEL to generate CD8aaþ T cells in vitro is

consistent with the hypothesis that CD82 IEL are the

immediate precursors of CD8aaþ IEL. Although CD82 T

cells were proposed previously to be precursors of

CD8aaþ IEL in H-Y TCR male mice, this has not been

directly demonstrated. One study suggested that immature

CD42, CD82 DN cells were exported from the thymus in

response to agonist peptide, despite their seemingly

immature phenotype.[40] Despite this, CD42, CD82 DN

cells were most likely “post-selection intermediates”

generated after co-receptor down-regulation in response to

agonist peptide. This is consistent with the presence of

CD8ab2=low; CD31low cells that accumulate in the

periphery of H-Y TCR male mice.[44] However,

CD8aaþ IEL develop in H-Y TCR male mice deficient

in the CD8bchain,[26] suggesting that high-avidity, CD8b-

mediated TCR signals are not be required for their

development. Moreover, the pattern of cytosine methyl-

ation in the CD8b gene locus of CD8aaþ IEL was not

consistent with the idea that CD8aaþ IEL are not the

progeny of auto-specific CD8abþ lineage cells that

escaped deletion in the thymus.[45]

The early and high level of TCR ab transgene

expression has been proposed as an alternative and

dominant mechanism “forcing” the accumulation of

CD42, CD82 DN cells in a “g-d like” pathway. These

cells, may home to the intestine and acquire CD8aa

expression in the intestine.[46,47] Although the generation

of CD42, CD82 DN precursors should not require the

agonist peptide in this case, the accumulation of CD8aaþ

IEL could still be dependent on the restricting MHC I

allele, H-2D b and the agonist peptide. We found that

CD82 IEL decreased and CD8aaþ IEL increased after

systemic treatment with TCR agonists but never to their

homeostatic maximum until late in postnatal life. In

addition, treatment with the cognate peptide failed to

increase CD8aaþ IEL in female mice (data not shown), as

it did in male mice in later postnatal life as we

demonstrated in this study. Therefore, although CD82

IEL could be generated by a similar mechanism in male

and female mice, peptide specific and agonist-dependent

events must pre-condition or cooperatively play a role in

the later generation and expansion of CD8aaþ IEL in

male mice. A similar mechanism may account for the

paucity of CD8aaþ IEL in early prenatal life male mice.

Perhaps the level of MHC-peptide or the density of APC is

not sufficient to activate or drive the differentiation of

CD8aaþ IEL from their precursors. Conversely, CD82

IEL may be intrinsically limited in their ability to generate

CD8aaþ IEL in early postnatal life when compared to

later in postnatal life.

The consistency in the ordered appearance of CD82

and CD8aaþ IEL during postnatal maturation in H-Y

TCR male mice with the appearance of these subsets in

mice with an unrestricted TCR repertoire is intriguing.[16]

Moreover, as several earlier studies showed that fetal and

early neonatal thymus was more efficient in the

generation of CD8aaþ IEL, than the thymus at later

times during life[13] perhaps precursors of IEL are

released during early events in the thymus and soon after

birth. Although our results are consistent with the

efficient generation of CD8aaþ thymocytes in fetal and

early neonatal thymic organ cultures[29,38,48] other cells in

the GALT and thymus must play a central role in the

kinetics of CD8aaþ IEL development. Whether it is as

simple for example, as the kinetics of non-classical MHC

class I expression, such as the thymus leukemia antigen

(TL) in the thymus versus the intestine,[48,49,50] or the

balance between signals derived from classical MHC I

molecules and their cognate peptide or the targeting of

distinct cell progenitors or APC to the intestine, remain to

be determined. Moreover, whether selection by non-

classical MHC I molecules enables male-specific

selection of CD8aaþ cells in H-Y TCR mice of

CD8aaþ IEL, similar to what has been those proposed

in other TCR transgenic systems[38,48,50] has not been

determined.

Defining the signals that restrict and then promote the

accumulation of CD8aaþ IEL during postnatal matu-

ration promises to elucidate not only key elements in this

developmental pathway, but a better understanding of self-

specific T cells as regulators in the prevention of

autoimmune diseases in the intestine and perhaps

elsewhere. Determining the origin of CD8aaþ IEL and

the requirements for their survival in the thymus of mice

with an unrestricted TCR will likely offer insight into

their in vivo role in intestinal immune homeostasis and

in disease. Thus developmentally regulated factors in

the thymus and intestine may carefully orchestrate

THE ONTOGENY OF CD8þ IEL 545

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the survival of potentially auto-reactive T cells for useful

responses in the prevention of autoimmunity not only in

the intestine, but perhaps in extra-intestinal sites as well.

Acknowledgements

We thank Mr. Joseph Thoits and Ms Kerstin Goth

(University of California, Irvine) and Ms. Erin Tobias for

technical assistance (University of Virginia), Ms. Joanne

Lannigan (University of Virginia) and Dr. Lisa Salazar for

cell sorting (University of California, Irvine).

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