T cell receptor antagonist peptides induce positive selection

Cell, Vol. 76, 17-27, January

14, 1994, Copyright

0 1994 by Cell Press

T Cell Receptor Antagonist Peptides Induce Positive Selection Kristin A. Hogquist,’ Stephen C. Jameson,” William R. Heath,t Jane L. Howard,* Michael J. Bevan,’ and Francis R. Carbone* *Howard Hughes Medical Institute Department of Immunology University of Washington Seattle, Washington 98195 fThe Walter and Eliza Hall Institute of Medical Research The Royal Melbourne Hospital Victoria 3050 Australia XIepartment of Pathology and Immunology Monash University Medical School Victoria 3181 Australia

Summary We have used organ culture of fetal thymic lobes from T cell receptor (TCR) transgenic j?&f(-I-) mice to study the role of peptides in positive selection. The TCR used was from a CD8+ T cell specific for ovalbumin 257-264 in the context of Kb. Several peptides with the ability to induce positive selection were identified. These peptide-selected thymocytes have the same phenotype as mature CD8+ T cells and can respond to antigen. Those peptides with the ability to induce positive selection were all variants of the antlgenic peptide and were identified as TCR antagonist peptides for this receptor. One peptide tested, El, induced positive selection on the /Mf(-l-) background but negative selection on the jMf(+/-) background. These results show that the process of positive selection is exquisitely peptide specific and sensitive to extremely low ligand density and support the notion that low efficacy ligands mediate positive selection. Introduction The T cell repertoire is shaped by the processes of positive and negative selection that occur in the thymus. Both result from the engagement of the T cell receptor (TCR) with a major histocompatibility complex (MHC) molecule plus peptide (Rothenberg, 1992). The former results in the survival of the cell and eventual differentiation into a functional lymphocyte; the latter results in activation-induced apoptotic death. The distinction of these two pathways is critical to understanding T cell development. The first suggestion of the role of peptides in positive selection came from studies of the effect of Class I MHC mutations on T cell selection (Sha et al., 1990; Nikolic iugic and Bevan, 1990; Jacobs et al., 1990). In these experiments, MHC molecules with mutations of residues in the peptide-binding groove were shown to influence positive selection either of a T cell response to antigenic peptide or of a TCR transgene. The mutations were predicted

to influence peptide binding (this was later demonstrated directly [Van Bleek and Nathenson, lSSl]) while leaving the TCR-facing residues intact, implicating groove-associated self-peptides in positive selection. Our previous work has established a model system with which to study positive selection of CD8+ T cells. This approach utilizes fetal thymic organ culture (FTOC) (Hogquist et al., 1993). Todate, FTOC istheonly invitro method for studying positive selection, since disrupted thymocytes do not undergo this developmental process. FTOC involves culturing thymic lobes from fetal mice at a gestational age of day 16. The lobes, which consist largely of double-negative immature thymocytes, are cultured under special conditions that allow diffusion of biological mediators such as peptides and antibodies into the lobe. Assessment of the phenotype of the cells over time in this system suggests that development in vitro mimics that in vivo (Jenkinson and Owen, 1990). The experimental approach we have used involves providing class I binding peptides to thymic lobe cultures to manipulate CD8+ T cell development. Because class I molecules with a large repertoire of self-peptides are present in lobes from normal animals, we have used thymic lobes from mice deficient in P2M. In these mice, heavy chains are not efficiently transported to the cell surface, because they do not form a complex with P2M and peptide. As a result, these mice profoundly lack CD8’ mature T cells (Koller et al., 1990; Zijlstraet al., 1990). Some empty heavy chains do traffic to the cell surface in cells lacking PPM, and T cells can recognize them if specific peptide and P2M are provided exogenously (Vitiello et al., 1990). This has provided a way to add back peptides and 8*M to assess thymic selection, and we have shown previously that this does restore CD8+ T cells (Hogquist et al., 1993). Our experiments as well as those using the TAP7(-I-) reconstitution system (Ashton-Rickardt et al., 1993) showed that self-peptides serve agreater function in positive selection than simply stabilizing MHC at the surface. Both of these experiments showed that complex mixtures of peptides are better at selecting the repertoire than single peptides when present at the same final concentration. This result led us to predict that positive selection would be highly specific, each TCR requiring a limited or even perhaps unique self-peptide-MHC complex for survival. To test this directly, we have applied the FTOC system to TCR transgenic mice. Using a class I restricted TCR of well-defined specificity on the /?ZA4(-/-) background, we were able to analyze the peptide requirement for positive selection of a single TCR. Results Thymocytes Expressing a TCR Transgene Specific for OVA,,/Kb Require the Kb Molecule for Positive Selection and Do Not Mature on a /IM(-l-) Background Transgenic mice were generated bearing aTCRwith spec-

Cell 18

ADULT THYMUS P2M -I-

p 2M +I-

Figure I. Positive Selection of Thymocytes Bearing the OVA-tcr-I Transgene Is Deficient in Mice That Lack b2M TCR transgenic thymocyies (from 5week-old mice) were stained for CD4, CD8, and Va2. The left panel shows the transaene SxDreSSed on an H-2b j&f(+/-) backgr&nd. The right panel shows it on an H-2b /&A+/-) background. The top panels show the CD4 versus CD8 profiles of total live thymocytes. The middle panels are histograms of Va2 expression indicating the gate area. The bottom panels show the CD4 versus CD8 profiles of thymocytes after gating on Va2”‘O” cells.

CD8 -

Va2 staining

ificity for an octamer peptide from ovalbumin (OVAZ-~,) in the context of the H-2Kb molecule. Expression of the transgene in thymocytes is shown in Figure 1. In H-2b b2M(+/-) mice, thymocytes show pronounced skewing to the CD8 single-positive (SP) population, compared to nontransgenic controls. Because rearrangement of the TCR u-chain locus is inefficiently blocked by allelic exclusion (in contrast with the P-chain), we analyzed the expression of the transgenic a-chain using an anti-Vu2 antibody(Gregoire et al., 1991). All of the cells express Vf35 (data not shown). By this means, we could show that the CD8 SP cells all expressed the transgenic receptor (Figure 1, bottom panel). This phenotype is similar to that seen in other Class I restricted TCR transgenic mice and is consistent with positive selection of the transgenic TCR into the CD8 SP subset (von Soehmer, 1990). The TCR transgenic mice were bred to H-2bj?2M(-I-) mice. The /3#(+/-) transgenics show a skewing toward CDSpositive cells among TCRhigh cells, as described above (20% of total thymocytes). However, the p2M(-I-) TCR transgenics (Figure 1, right panels) lack this population (0.1%). The overall defect in positive selection of the transgene can also be seen in the Vu2 staining profile (Figure 1, middle panel). While the /3&(+/-) transgenics show 69% TCRhighcells in the thymus, thep2/U(-/-) thymocytes express predominantly intermediate levels of TCR

(8% TCRhigh). The absence of CD8+ Va2”‘gh cells in the spleen of the p2hn(-/-) mouse was also noted (data not shown). To identify the Class I molecule responsible for selection of this receptor, H-2b transgenic animals were bred to H-2bm1mice (Figure 2). TCRh@Jh thymocytes from H-2bm’ homozygous transgenics show very poor skewing to the CD4-8+ subset and have much lower levels of transgenic TCR expression compared with the H-2b mice (Figure 2) while in H-2b x bml transgenic animals, expression of the TCR transgene in the thymus is similar to that observed in H-2b mice. These findings indicate that the lack of TCRhighCD8 SP in the H-2bm1animals is not due to deletion of cells bearing this receptor (which would be apparent in the H-2b x bmlanimals), but rather that H-2bm1is incapable of supporting positive selection of this receptor into the CD8 SP compartment. H-2bm’mice express the same MHC molecules as H-2b animals except for Kbm’, which differs from Kb by three amino acids. These mutations are known to alter peptide binding, T cell recognition, and positive selection (Joyceet al., 1991; Shaet al., 1990; Nikolicfugic and Sevan, 1990). These data indicate that Kb is responsible for positive selection of the transgenic TCR into the CD8 SP compartment. The few CD4 SP TCRhighcells seen on nonselecting backgrounds (and to some degree on selecting backgrounds) possibly arise via rearrangement of

Peptide Requirement 19

for T Cell Selection

ADULT THYMUS B6xbml

Figure 2. The OVA-tcr-I TCR Requires Kb for Positive Selection In Vivo TCR transgenic thymocytes (from B-week-old mice) on the three different strain backgrounds indicated were stained for CD4, CD6, and Va2. The top panel shows the CD4 versus CD6 profiles of thymocytes after gating on VaZ”‘* cells. The bottom panel shows a histogram of Va2 staining and an indication of the gate area.

bml

Fl

Va2 staining

endogenous a-chains that are coexpressed with the transgenic a- and @chains and enable positive selection on class II. Antigenic Peptide Causes in FTOC from Both B#(+I-) To study the role of peptide performed with thymic lobes gestational age day 16. After

Negative Selection and fi&f(-l-) Mice in development, FTOC was taken from OVA-tcr-I mice at 7 days in culture, thymocytes

developed a phenotype similar to the adult (Figure 3, top). The skewing of the transgene on a /3,M(+I-) background to CD8 was striking (55% of the thymocytes were CD8’ Va2”“J”). On the &U(-I-) background, this was severely reduced (3%). While the percentage of CD8 mature T cells in bZM(-l-) FTOC was slightly higher than in the adult (3% versus 0.10/o), this is still 20-fold lower than on a &M(+/-) background. When 20 uM antigenic peptide is added to these cultures

FTOC

Figure 3. FTOC with the Antigenic Peptide Leads to Deletion in Both p&f(+/-) and b&f (-I-) Strains

-I- lobes

+I- lobes P i ! I!

A., ,:

NONE

e.:..,

R

+OVAp

FTOC was performed with thymic lobes from BN(+/-) and /3&f(-I-) TCR transgenic mice at a gestational age of day 16. Cultures contained 5 pg/ml human t&M alone (top panels) or plus 20 uM OVA, peptide (bottom panels). Media, including peptide and B2M, were replenished daily. After 7 days of culture, the thymocytes from these lobes were stained for CD4, CD6, and Va2. This figure shows the CD4 versus CD6 profiles after gating on Va2”@ cells.

-

100 -

90

---o--

E I

70 1

80-

60

---.--

--a--

---.----A-.A

40k D ax polySER K4 R4 V-OVA P7 Kl El

O ”Ap FLU

..&/f/

50-I .... 10-l’ ~~~~~1”‘~~~~~~~~~o~.4

00 none

100 pM 3.3 nM

OVAp Figure 4. Decreased Efficiency TCR Transgenic Lobes

100 nM

3.3 uM

100 UM

concentration of Thymocyte

Figure 5. The Peptides Used All Bind and Stabilize KD Deletion in /3&(-I-)

FTOC was performed as described in Figure 3, except that various concentrations of the peptide OVA, were used. Cells were counted and stained after 7 days. The number of CD4, CD8 DP cells remaining in each lobe was determined by multiplying the number of cells recovered by the percentage that were positive for both CD4 and CD8. The data are the average of two to three lobes in each group. The yield in the peptide-untreated group was considered 1.0, and the other groups are expressed relative to it.

along with an exogenous source of p,M, a massive deletion occurs, with predominantly double-negative cells remaining. This happens whether the lobes are &IJ(+/-) or (-I-). This was predicted by our previous demonstration that peptide can target /I&!(-I-) cells for lysis but that targeting was 50-fold less efficient compared with &B2M(+/-) targets (Hogquist et al., 1993). A similar shift in the peptide dose response was seen in organ culture (Figure 4). To determine the extent of negative selection, the number of double positive cells remaining in the culture was calculated after a 7 day organ culture with various doses of the antigenic peptide. About 50-fold more peptide was required to achieve maximal deletion in pfi(-I-) lobes, as compared with pN(+/-) lobes. The above experiments demonstrate that the low levels of class I heavy chains that traffic to the cell surface in &A@/-) cells can be stabilized by the addition of class I binding peptides plus P2M and that these complexes can affect thymic selection of the transgene. Although we have never been able to detect these increased complexes on the cell surface by staining with anti-class I antibodies (data not shown), the T cell is clearly quite sensitive to these low levels. Peptides Induce CD8 Development in /3M-l-) Mice Various peptides, all of which bind and stabilize Kb, were next tested for their ability to induce positive selection in FTOC of the pN(-/-) mice. Three types of peptides were tested: first, natural self-peptides; second, a null peptide; and third, variants of the antigenic peptide. One hypothesis is that the most abundant self-peptides present on thymic epithelial cells mediate positive selection. The naturally occurring self-peptides we used to test this were identified by direct sequencing from Kb immunoprecipi-

The RMA-S stabilization assay was used to detect peptide binding to KD. Cells were incubated in various concentrations of peptide for 4.5 hr, after which the level of K” on the surface was determined by FACS analysis. The mean fluorescence intensity is shown.

tates (S. Imaeda, unpublished data). These peptides were found to have homology to a yeast CDC protein and a bacterial 40 kd ribosomal protein. What we have termed a null peptide is a polyserine octamer peptide (polySER). This peptide has serine at positions 1, 2, 4, 6, and 7, four of which are the predicted TCR contact residues in a Kb binding peptide. At the other positions are the Kb anchor residues. This peptide binds Kb well but does not have lengthy solvent-exposed side chains. The polySER peptide was used to test the hypothesis that any peptide that would bind Kb well but not prevent the TCR from interacting with the MHC residues would be a good positively selecting peptide. Finally, variants of the antigenic peptide that have decreased or no ability to stimulate mature cytotoxic T lymphocytes (CTL) via this particular TCR were used to test what has loosely been termed the affinity hypothesis of positive selection. All peptides used were similar in their ability to bind Kb (within 20-fold of OVAp control), as determined by the RMA-S stabilization assay (Figure 5). A Db-binding peptide from the influenza nucleoprotein was included as a negative control. Since thymocyte deletion occurred readily on both backgrounds, the peptides were prescreened using an in vitro deletion assay with transgenic thymocytes (Swat et al., 1991). Only those peptides that caused little or no deletion in this assay were included (data not shown). For FTOC, peptides were used at a final concentration of 20 PM, in the presence of exogenous B2M. Figure 6 shows fetal thymocytes after 7 days of treatment with three of these peptides. The antigenic peptide variants, V-OVA and El, were able to induce CD8’ T cell development in the p,M-/- lobes (Figure 6, left panel). El was complete in its effect, while V-OVA was less so. In /&#(-I-) lobes to which no peptide was added, the percentage of cells that are TCRhigh CD8 SP is 5.2 I?: 3.4. Upon incubation with V-OVA, this increases to 16.3 + 5.1; with El it is 45.2 -c 8.0. The El-treated cultures have a profile that is very similar to that of cells selected in /3N(+/-) lobes, which have 48.2% f 13% TCRhigh CD8 SP cells. This effect can also be seen in the overall levels of TCR ex-

Peptide Requirement 21

for T Cell Selection

P2M -I-

Figure 6. Variants of the Antigenic Mediate Positive Selection

FTOC was performed as described in Figure 3, using 20 uM of each of the three Kb binding peptides, polySER, V-OVA, and El (sequences are shown in Table l), or no peptide. The left and right panels show the CD4 versus CD8 profiles from &Lf(-l-) or jN(+I-) lobes, respectively, after gating on Va2”m ” cells. In the middle panel are histograms of Vu2 staining from BM(-I-) lobes. Data shown are representative of two to five experiments.

1 i----I !: I-

;; u CDS

Va2 staining

Peptide

CDS

pressed on thymocytes in j%M(-I-) FTOC (Figure 6, middle panels). Two other OVAp variants, R4 and Ki , also induced CD6 differentiation to various degrees (Figure 7) while polySER as well as the naturally occurring selfpeptides had no effect (Figures 6 and 7). These CD6+ cells have the same phenotype as CD8’ cells that mature in the p2M(+/-) mouse. They express high levels of the transgenic receptor (see Figure 6). Positively selected thymocytes differ from immature thymocytes in that they have down-regulated expression of heat stable antigen (HSA) (Nikolic fugic, 1991) and up-regulated expression of Bcl-2 (Veis et al., 1993). The El-selected ceils, like those selected on the wild-type background, are also HSA-negative and express Bcl-2 (Figure 8). Because a low level of CD8 SP cells is present after 7 days in untreated/?&f(-I-) FTOC, it was possible that the El peptide was simply expanding this population. If this were so, the double-positive (DP) cells would remain unchanged. This is not the case. The DP cells decrease in absolute numbers as well as percentage, while CD8 SPs increase, suggesting differentiation. In addition, the El-selected cells do not proliferate in response to El, even in the presence of EL4 antigen-presenting cells (data not shown).

indicate that this is not the case. Lobes were cultured for 7 days with various peptides, then disrupted and washed. Fluorescence-activated cell sorting (FACS) analysis was performed on half the cells. The other half was placed in culture with EL4 antigen-presenting cells, with or without 10 nM OVAp peptide. Thymocyte input was normalized as to the number of lobes the cells came from, such that the proliferation would represent the overall activity developing under those conditions. While @+I-) lobes cultured with P2M alone had very little ability to respond to antigen, when El or V-OVA were included in the organ cultures, the developing cells were able to respond vigor-

P2M +/- 1 NONE El R4 Kl P2M

-/-

V-OVA P7 polySER “self” NONE

Peptide-Selected Cells Respond to Antigen Despite their phenotypic similarity to cells positively selected on a p&f(+/-) background, it was possible that the cells developing in the presence of variants of the antigenie peptide were functionally anergic. That peptide variants can cause anergy in mature CD4 T cell clones was recently shown (Sloan Lancaster et al., 1993). The results of a proliferation assay with FTOC thymocytes (Figure 9A)

0

10

20

30

40

50

60

% CD8+ TCRhi cells Figure 7. The Effect of Various Peptides on Positive Selection FTOC was performed as described for Figure 6, using 20 uM of each of the peptides listed (sequences are shown in Table 1). The percentage of cells that are CD6 SP, Va2”‘@ ’is shown. Lobes were analyzed individually, and the mean was determined. Error bars indicate the standard error of the mean. Data are from 4-16 lobes in each group.

Cell 22

B&2

HSA

Figure 8. The Phenotype of Peptide-Selected CD8 SP Cells Is the Same as That of Wild-TypeSelected Cells FTOC was performed as described in Figure 3, using 20 pM El peptide. Cells were stained for CD4, CD8, and HSA or B&2. The histograms show the HSA or Bcl-2 expression on CD4, CD8 DP cells (dotted line) and CD8 SP cells (solid line).

P2M-/NONE

P2M-IEl

P2M+/NONE

-

-

-

CD4,CD8 DP CD8 SP

ously to antigen. The cells that responded were analyzed by FACS and were CD8 SP (data not shown). In agreement with the data shown in Figure 3, lobes incubated in OVAp underwent deletion; the remaining cells were unable to respond to this same peptide after the 7 days of organ culture. Ligand Density Can Affect Thymic Development It is interesting to note the different effect of the El peptide on p#(+/-) versus /&y-I-) transgenic thymic lobe cultures. El in the b2M(-/-) lobes is clearly inducing a differentiation of T cells that at this point is indistinguishable from positive selection in /?&I(+/-) FTOC. El in the p# (+I-) lobes, on the other hand, has a clearly negative effect. After 7 days in FTOC, the remaining cells are either double-negative or CD8’O ”(Figure 6, right panel) and have an impaired ability to respond to antigen compared with that of untreated lobes (Figure 9B). We interpret this phenotype to be a partial deletion, since a similar effect is seen at low OVAp concentrations in /?&f(+I-) lobes (data not shown). Cells lacking f3*M are approximately 50-fold less efficient at presenting antigen to T cells. The difference between the +I- and the -I- effect therefore probably lies in the density of El-@ complexes presented to the thymocyte. Positively Selecting Peptides Are TCR Antagonists A positively selecting ligand is predicted to be one that could engage the TCR without leading to a typical activation signal. The definition fits that of a recently described

/GM-/- lobes

NiNE

B

200

V-&A

E;

0; A

p2M+/- lobes

+ OVAp

• Zl

NONE

160

2

120

i ”

80

40 0 N&NE

V-&A

E’1

0; A

peptide added to FTOC Figure 9. Peptide Variant-Selected genie OVAp Peptide

Thymocytes

Respond to the Anti-

Cells from organ culture, to which 20 uM of the peptides V-OVA, El, OVAp, or none had been added, were harvested and counted. A fraction of the cells from each lobe was stained as in Figure 3. The remaining cells from four lobes in each group (except V-OVA, which had three) were added to cultures with EL4 antigen-presenting cells with or without 10 nM OVAp. PHlthymidine incorporation was determined after 48 hr.

Peptide Requirement 23

Table 1. Summary

for T Cell Selection

of the Effects of Various Peptides in FTOC and in CTL Lysis Assays Thymic Selection

Peptide

Sequence

40 kd CDC PolySER P7 V-OVA Kl R4 El OVAp

/%M-l-

GAYEFTTL ASYEFTQL SSYSYSSL SI INFEPL RGYNYEKL KI INFEKL SI IRFEKL El INFEKL SI INFEKL

None None None Weak Positive Positive Positive Positive Positive Negative

Lysis Assay

ri

-1,

-10

-9

-8

[Peptide]

-7

-6

-5

a-e + + -a-

40 KD CDC PolySER P7 OVAp

-

+I-

+ + + + + -

-nt-a-+)-a--

V-OVA Kl R4 El OVAp

-4

-12

-11

-10

-9

Lysis Assay

j

:

[Peptide]

(loglo M)

it+++

In this paper, we show that single peptides, which are variants of the antigenic peptide, can induce positive se-

[Peptide] Prolonged

+I-

Discussion

30 -:

None None None None None Negative None Negative Negative

Figure 10. Agonist and Antagonistic ties of the Peptides Used in FTOC

Assay

Proper-

The capacity of various peptides to induce or antagonize lysis by a CTL line derived from a transgenic mouse was tested in a “Cr release assay. (A), direct lysis assay (4 hr); (B), prepulse antagonist assay; (C), direct lysis assay (5 hr). For the antagonist assay, target cells were prepulsed with 2 pM OVAp. Specific lysis in the absence of added peptides was 37%. The data in (B) show percent inhibition of this lysis in the presence of the indicated peptides. (A) and (B) were from the same experiment, (C) from a second experiment.

(loglo M) C)

Antagonist

-

B) Antagonist

I r/

-12

Agonist

the response to antigenic peptide at intermediate concentrations, but at high concentrations directly stimulated the TCR. Within the small set of peptides we have tested in theseexperiments, it appearsthat those that mediate positive selection are antagonists in the prep&e assay. However, the degree to which a peptide causes positive selection is not predicted by its potency as an antagonist. For example, P7 is a strong antagonist (Figure lOB), yet is relatively weak at inducing positive selection.

class of peptides called TCR antagonist peptides (De Magistris et al., 1992). Table 1 shows that, in fact, those peptides we have identified that caused CD8 development in FTOC belong to this class. Antagonist peptides for this TCR were identified using a prepulse assay for inhibition of CTL lysis (Jameson et al., 1993). Figure 10 shows a dose response curve of each of the peptides for their ability to stimulate this TCR directly (Figures 10A and 1OC) as well as for their ability to antagonize the response to low doses of OVAp (Figure 1OB). Some peptides, such as V-OVA and R4, are strict antagonists. These peptides do not stimulate the T cell directly, as far as we can tell using CTL lysis as a readout. Other peptides, such as El and Kl , are agonist/antagonists. The Kl peptide antagonized

A)

&M+I-

+CDC + +

P7 V-OVA

--t -a-a-

Kl R4 El

-8

-I

-6

-5

(loglo M)

-4

lection in /32M(-I-) TCR transgenics. Because thymocytes from these mice have a single specificity, the effect on selection is very dramatic. The cells selected on variant peptides are indistinguishable from the cells selected on afi,M(+/-) background (which provides a natural selecting ligand). These cells are CD4 negative, CD8 positive and express high levels of the transgenic a-chain. They are distinct from immature thymocytes in that they express low surface levels of HSA and have Bcl-2 protein in the cytoplasm. The early activation antigen CD69, which has been shown to be expressed upon positive selection in the thymus (Swat et al., 1993; Bendelac et al., 1992), was not present on the peptide-selected cells in our system. However, the cells that have matured in a p#(+/-) lobe are also CD69-negative. Thymocytes that mature in FTOC are known to develop as a cohort over time. Since CD69 is expressed immediately after activation, then downregulated, it is likely that CD69 may have been expressed earlier in the lobes and been shut off by the day of the assay. The kinetics of expression of various gene products during peptide induction of selection is currently being investigated. In addition to phenotypically resembling the wild-type positively selected cell, the peptide-selected cells responded to the antigenic peptide. Thus, we have shown that the addition of single synthetic peptides to FTOC induces a differentiation pathway leading not only to survival but also to functional maturation. Peptide Specificity Our previous work led us to propose that T cell repertoire selection requires acomplex mixtureof peptides, because each single TCR requires a specific self-peptide-MHC ligand. An alternative hypothesis is that immature T cells need only interact with MHC residues, but that peptides can have a negative effect on this process. In other words, a peptide with a long or bulky side chain extending out of the groove might block TCR engagement with the MHC and so prevent positive selection. The polyserine peptide we have used is one that, based on the crystal structure of the peptide-Kb complex, would not have side chains extending out of the groove. If the hypothesis is true that peptides need only stay out of the way, this type of peptide should select a Kb-restricted TCR. However, the polySER peptide had no effect on thymocyte differentiation of the OVA-specific TCR in these experiments. Two peptides that are abundant naturally occurring self-peptides presented by Kb also had no effect. The peptides that were able to mediate selection of this receptor are all variants of the antigenic peptide (OVAp). Of the four peptides identified that caused positive selection, three are single amino acid variants. A single amino acid change (arginine at position 4, for example) caused the loss of negative selection, yet stimulated positive selection. Since the side chains that affect selection are those that extend up and out of the groove, it seems likely that the peptides are directly participating in the specificity of positive selection. Given this level of specificity, it is surprising that a single allele-binding peptide selected a large number of CD8+ T cells in FTOC of TAP7(-I-), nontransgenie mice (Ashton-Rickardt et al., 1993). This same peptide

had only asmall effect in the nontransgenic@U(-l-) system (Hogquist et al., 1993). A major difference between these two systems is the level of binding achieved when peptide is added. In TAP7(-I-) cells, single peptides can bind empty heavy chains and stabilize MHC to levels similar to a wild-type cell, as detected by FACS analysis. In pZnn (-I--) cells, no increase can be seen by FACS analysis. There are, then, many times more single peptide-MHC complexes on the surface in TAP7(-I-) cells compared with b&f(-I-) cells. In normal cells, the MHC molecules present a vast array of different self-peptides, predominantly derived from the intracytoplasmic pool of proteins. The average representation of asingle peptide-MHC complex is around 0.1% of the total complexes per cell (Hunt et al., 1992). Therefore, the density of single peptide-MHC complexes on /3#(--I-) cells that have been provided synthetic peptide and j3,M is probably closer to the physiologic range than in TAP7(-I-) cells provided with the same amount of peptide. It is possible that overrepresentation of a single peptide-MHC complex on TAP7(-I-) cells led to differentiation of T cells with specificities that normally are not selected. Many single amino acid variants of OVAp that will stimulate transgenic T cells will delete thymocytes bearing this receptor in an in vitro deletion assay and in FTOC (data not shown). The variants that mediate positive selection are all extremely weak (requiring peptide concentrations in the PM range; greater than 7 logs less active than the antigenic peptide) or negative in their ability to target EL4 cells for lysis by CTL bearing this receptor. Despite their weakabilitytostimulateCTL, we knowthatthesepeptides, when bound to Kb, can efficiently engage the TCR, since they act as antagonists in the low nanomolar range. All of the positively selecting peptides identified here are antagonists for mature CTL expressing this receptor. It should be pointed out that antagonist peptides have an inhibitory effect only within a very small range of antigenic peptide concentration and only when the antagonist peptide is present in considerable excess (Jameson et al., 1993; De Magistris et al., 1992). Therefore, it is unlikely that expression of an endogenous antagonist peptide in the periphery would inhibit a T cell response.

Implications for the Mechanism of Positive Selection That TCR antagonist peptides would exist and that they would mediate positive selection was predicted several years ago (Mannie, 1991). In 1992, De Magistris et al. first demonstrated TCR antagonism with CD4+ T cells. More recently, we defined the pattern of TCR antagonism of three CD8+ clones specific for OVAp plus H-2Kb, using a panel of single amino acid variants of the antigenic peptide (Jameson et al., 1993). There are two models to explain TCR antagonism. It has been proposed that the affinity of the TCR for antagonist-MHC complex may be very weak compared with affinity for the agonist complex (Alexander et al., 1993). If cross-linked complexes of MHC-TCR recognition units have to form for efficient TCR signal transduction, then the presence of low affinity ligands on

Peptide Requirement 25

for T Cell Selection

the antigen-presenting cell may generate unstable assemblies and disrupt signaling. An alternative model is independent of affinity concerns, and holds that antagonist peptides would engage the TCR without inducing aconformational change critical for signal transduction (Mannie, 1991; Janeway, 1993). Further analysis with peptide variants, as well as defining the affinities of the various peptide/Kb complexes for the TCR, will be required to distinguish between these models. Both models, however, raise the possibility that the TCR does not directly transduce a signal either during a response to antagonist or, likewise, during positive selection. Rather, its receptor may simply hold the T cell in contact with a thymic epithelial cell while other molecules (accessory or adhesion molecules) would transduce the signal for survival. There are, however, data that some antagonist peptidevariantscan induce differential signaling in mature T cells and that others can induce anergy (Evavold et al., 1993; Sloan Lancaster et al., 1993; Racioppi et al., 1993). This suggests that antagonists deliver a signal through the TCR that is distinct from that provided by the antigenic peptide, and this may also be true for positively selecting ligands. The El peptide-MHC ligand has a given affinity for the transgenic TCR. The fact that the El peptide can cause positive selection when present at low ligand density (P,M(-I-)) and partial negative selection when present at high ligand density &M(+/-)) rules out a sole determinative role for affinity in thymic selection. We use the term efficacy to refer to the signaling property of ligands in activation of mature T cells or in deletion of thymocytes, noting that it includes the contribution of the TCR as well as accessory molecules (CD4, CD8, and other molecules). Enhanced affinity or avidity of the TCR for MHC as well as factors such as increased CDB-MHC ligand interactions could lead to increased efficacy. Several experiments suggested that altering the CD8-MHC interaction alone can affect selection (Robey et al., 1992; Sherman et al., 1992; Lee et al., 1992). Our results support a model where low efficacy ligands mediate positive selection. For most TCR specificities, it is assumed that the selfpeptide that can select the receptor is distinct from the foreign antigenic peptide. However, the El result indicates that, at least in some cases, receptors might be selected on their antigenic peptide ligands at low density. We have not seen this with OVAp in this transgenic system (data not shown), although it is possible that the increment between doses tested in our experiments (see Figure 4) was not small enough to have revealed positive selection. Additionally, enhanced positive selection at low antigen concentration was not seen in FTOC with a Class II restricted receptor (Spain and Berg, 1992). Our observation that the OVA-tcr-I Tcell receptor, which was selected in a normal thymus, can be selected in vitro upon interaction with only certain peptides and at the low levels of peptide/Kb that are present on the surface of p&f(--I-) cells argues that positive selection is highly specific and sensitive. In these experiments, we have defined a system in which peptide can be added to induce positive selection of a single T cell receptor. This will allow us to investigate further the biochemical pathway and subse-

quent gene expression that result from this step in thymocyte differentiation. Experimental

Procedures

Transgenic Mice The TCR transgenic animals referred to as OVA-tcr-I express the Va2, V65 TCR derived from the p-restricted OVA257-254-specific CTL clone 149.42 (Kelly et al., 1993). This T cell uses rearranged Va2Ja26 and Vf35-D82-Jf32.6 TCR a- and b-chains, respectively. The cDNA encoding the complete 149.42 a-chain was cloned into the pES4 expression vector (Kaye et al., 1992) and coinjected into blastocysts of (66 x bml)Fl mice, together with the genomic B-chain construct, pKS913. CB16.31 (Carbone et al., 1992). Transgenic founders 243-2 and 253-2 were identified and have similar expression of transgenes (data not shown). Progeny of line 243-2, backcrossed to B6 mice, were used for the experiments described here. Peptides Peptides were synthesized at the Howard Hughes Chemical Synthesis Facility (University of Washington, Seattle, Washington) or using an Applied Eiosystems (Foster City, California) Synergy peptide synthesizer.

FTOC Female &M(-I-) mice were mated to male /3&f(+/-), TCR transgenic mice. Thymic lobes were excised from mice at a gestational age of day 16 (the day of the plug was considered day 1). The lobes were put on cellulose ester filters (Millipore, Bedford, Massachusetts) that were placed upon gelfoam sponges (Upjohn, Kalamazoo, Michigan) in media with or without peptide (used at 20 uM unless otherwise indicated). The media consisted of RPM plus 10% fetal calf serum, penicillin, streptomycin, L-glutamine, P-mercaptoethanol, and 5 Kg/ml human P2M (Calbiochem, La Jolla, California). Media were replenished each 24 hr. After 7 days, thymocytes were released from the lobes by pressing through a steel mesh. Flow Cytometry The anti-p antibody Y3 (American Type Culture Collection, Rockville, Maryland) was used to type mice as 6&l + or -. The anti-Va2 antibody 620.1.1 was used for TCR transgenic typing. Anti-CD4 phycoerythrin and anti-CD8 fluorescein isothiocyanate (Becton-Dickinson, Mountainview, California) were used directly. Antibodies to Va2 (gift of P. Fink), HSA, and CD69 (Pharmingen, San Diego, California)were biotinylated. A hamster antibody to mouse Bcl-2 (Veis et al., 1993) (gift of D. Hockenberry and S. Korsmeyer) was used, followed by biotin goat anti-hamster immunoglobulin (Vector, Burlingame, California). Tricolor streptavidin (Caltag, San Francisco, California) was used as a secondary reagent. For class I staining, Y3 was used, followed by fluorescein isothiocyanate goat anti-mouse IgG (Cappel, Malvern, Pennsylvania). For detection of B&2, which is present in the cytosol, a saponin solubilization assay was used (Veis et al., 1993). Cells were analyzed on a FACScan. CTL Lysis Assays A CTL line was established from C57BU8 OVA-tcr-I mice. Briefly, splenocytes were stimulated with 100 pM OVAp for 14 days and subsequently restimulated weekly with E. G7 (EL4 transfected with the ovalbumin gene [Moore et al., 19881) in the presence of rat concanavalin A supernatant. In experiments to determine antagonist activity, a prepulse assay was used as described (Jameson et al., 1993). EL4 cells ware labeled with [SCr]sodium chromate in RPIO for 1 hr at 37%. A suboptimal concentration of OVAp (2 PM) was introduced for the duration of the labeling. The cells were washed three times, and l(r cells were transferred to each well of a 96 well plate. Test peptides were added at various concentrations. After 30 min at 37°C 3 x 1 O4CTL were added per well. After 4 hr, the plate was centrifuged and 100 ul of the supernatant removed and counted. In experiments to determine direct lysis, labeling was performed in the absence of OVAp and lysis determined at 4 or 5 hr as indicated.

Proliferation Aeeey Fetal lobes after 7 days of in vitro culture were disrupted by pressing through a steel mesh. Ceils from the lobes were washed three times and added to a 96 well plate with 3 x l(r irradiated EL4 antigenpresenting cells with or without 10 nM OVAp. After 46 hr at 37% 0.5 uCi per well of [%]thymidine (New England Nuclear Research Products, Boston, Massachusetts) was added. After an additional 6 hr at 37%, the cells were harvested and the incorporated radioactivity measured.

RMA-S Stabilization Assay To detect binding of peptides to Kb, we used the RMA-S stabilization assay (Schumacher et al., 1990). Briefly, RMA-S cells were incubated at 31 OC overnight to provide maximum MHC expression at the cell surface. Peptide was added at various concentrations for 30 min at 31 OC. Cultures were shifted to 37% for 4 hr. Ceils were then washed and stained for Kb expression as described above.

Acknowledgments Address correspondence to M. J. B. The authors wish to thank Kim McConnell for help with the mouse breeding, Stan Korsmeyer and David Hockenberry for providing the antibody to mouse Bcl-2, Sasha Rudensky for use of the Synergy peptide synthesizer, and Jon Kaye for the pES4 vector. We are grateful to Suguru lmaeda for providing information prior to publication, and to Stacey Dillon, Michael Starnbath, and Sasha Rudensky for critically reading the manuscript. This research was supported by the Howard Hughes Medical Institute and by the National Institutes of Health (Al-28902). F. R. C. is supported byfundsfrom the Australian ResearchCouncil, the Australian National Health and Medical Research Council, and a Cancer Research Institute investigator award. S. C. J. is a special fellow of the Leukemia Society of America. W. R. H. was funded by a fellowship from the Australian Research Council. Received

October

16, 1993.

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