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A Model for the Origin of TCR-ß⁺ CD4^-CD8^- B220⁺ Cells Based on High Affinity TCR Signals¹

Immunobiology Program, Department of Medicine, University of Vermont College of Medicine, Burlington, VT 05405

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The origin of TCR-ß⁺ CD4^-CD8^- cells is unclear, yet accumulating evidence suggests that they do not represent merely a default pathway of unselected thymocytes. Rather, they arise by active selection as evidenced by their absence in mice lacking expression of class I MHC. TCR-ß⁺ CD4^-CD8^- cells also preferentially accumulate in mice lacking expression of Fas/APO-1/CD95 (lpr) or Fas-ligand (gld), suggesting that this subset might represent a subpopulation destined for apoptosis in normal mice. Findings from mice bearing a self-reactive TCR transgene support this view. In the current study we observe that in normal mice, TCR-ß⁺ CD4^-CD8^- thymocytes contain a high proportion of cells undergoing apoptosis. The apoptotic subpopulation is further identified by its expression of B220 and IL2Rß and the absence of surface CD2. The CD4^-CD8^- B220⁺ phenotype is also enriched in T cells that recognize endogenous retroviral superantigens, and can be induced in TCR transgenic mice using peptide/MHC complexes that bear high affinity, but not low affinity, for TCR. A model is presented whereby the TCR-ß⁺ CD2^- CD4^-CD8^- B220⁺ phenotype arises from high intensity TCR signals. This model is broadly applicable to developing thymocytes as well as mature peripheral T cells and may represent the phenotype of self-reactive T cells that are increased in certain autoimmune conditions.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
TCR-ß⁺ CD4^-CD8^- (CD4^-8^-)⁴ T cells represent an unusual and minor phenotype in most mouse strains, but accumulate to great numbers in mice bearing mutations in the genes for fas (lpr) or fas-ligand (gld) (1, 2, 3). In most normal mouse strains TCR-ß⁺ CD4^-8^- cells contain a subpopulation of NK1⁺ T cells that manifest a highly skewed TCR bearing an invariant -chain, V14-J281, paired with either Vß8.2, Vß7, or Vß2, and recognize the class I-like MHC molecule, CD1 (4, 5, 6, 7, 8). It remains an enigma to what extent the signals that give rise to CD4^-8^- T cells are similar among the total group of CD4^-8^- T cells in normal mice, the V14⁺ NK1⁺ subset, and the CD4^-8^- T cells in lpr mice (9, 10). Mounting evidence suggests that TCR-ß⁺ CD4^-8^- cells do not represent a residual immature subset arising from an unselected default pathway, but rather arise by an active selection process. This result is supported by the findings that in both normal and lpr mice the loss of class I MHC expression results in the near absence of TCR-ß⁺ CD4^-8^- cells (11, 12, 13, 14). In addition, this subset in both strains of mice has likely previously expressed CD8, as evidenced by persistent demethylation of the CD8 gene (15, 16), further supporting their common origin by positive selection, primarily by class I or class I-like MHC molecules.

The emergence of TCR-ß⁺ CD4^-8^- T cells also has been incidentally observed to various degrees in fetal thymic organ cultures containing peptide variants of the native Ag (17), as well as on lymphocytes that appear in the liver following Ag exposure (18). A similar CD4^lowCD8^low phenotype also appears on unmanipulated dying thymocytes during short-term in vitro culture (19). CD44 and CD69 expression was observed on this apoptotic subset, although expression of these activation markers was not confined to this subset. These additional findings further suggest that CD4^-8^- T cells arise from active signaling.

Additional examples of TCR-ß⁺ CD4^-8^- T cells occur in mice bearing a transgenic TCR that is self-reactive, such as in male H-Y-reactive TCR transgenic mice (20), and two independent K^b-reactive TCR transgenes when expressed in C57BL/6 mice (21, 22). The accumulated observations led us to propose a model for the origin of TCR-ß⁺ CD4^-8^- cells as resulting from a high intensity signal delivered by the TCR and associated costimulatory molecules (23). This subset was consequently predicted to be on the verge of negative selection, if not actively undergoing apoptosis. Lower intensity TCR signals would result in a phenotype more typical of mature CD4⁺ and CD8⁺ T cells with less apoptosis.

To directly test predictions of this model, we initially observed increased B220 and IL2Rß, and decreased CD2 as the surface markers that most reliably identified the apoptotic subset of thymocytes. In vivo systems were then used to identify peripheral T cells reactive to the endogenous superantigen, mouse mammary tumor virus (MTV), as being enriched within the CD4^-8^- subset, many of which express B220. Finally, two different TCR transgenic mice that each react to chicken OVA, one restricted to class I MHC and the other to class II MHC, were used to demonstrate that the TCR-ß⁺ CD2^- CD4^-8^- B220⁺ phenotype can be induced in vivo most effectively by high affinity OVA peptides, whereas lower affinity OVA peptide variants caused less apoptosis and more expansion of mature CD4⁺ or CD8⁺ T cell phenotypes. The findings suggest that the TCR-ß⁺ CD4^-8^- subset identifies a population enriched for self-reactive T cells that can be increased in certain autoimmune disorders (24).

	Materials and Methods

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Mice

Normal strains of C57BL/6, BALB/c, and MRL^+/+ mice were bred at the animal facilities of the University of Vermont College of Medicine from original breeding pairs obtained from The Jackson Laboratory (Bar Harbor, ME). DO.11.10 TCR transgenic mice recognize chicken OVA peptide 323–339 (OVA_II) in the context of class II MHC I-A^d, and were the kind gift of Dr. Dennis Loh (25). DO.11.10 mice were maintained by breeding transgenic male mice to normal BALB/c females. Offspring bearing the TCR transgene were identified by expression of the clonotype TCR identified by mAb KJ1-26. OT-1 mice recognize chicken OVA peptide 257–264 (OVA_I) restricted to class I MHC, K^b, and were kindly provided by Drs. Francis Carbone and Michael Bevan (26). OT-1 mice were maintained by breeding TCR transgenic male mice to normal C57BL/6 females. Offspring were screened for the clonotype TCR using anti-V2 mAb.

Abs, cell preparations, and flow cytometry

Monoclonal anti-murine CD8 conjugated to PE, CD44-FITC, and B220-PE were purchased from Caltag (Burlingame, CA). Monoclonal anti-murine CD4 conjugated to Red613 was purchased from Life Technologies (Gaithersburg, MD). Monoclonal anti-murine V2 conjugated to FITC or PE, CD69-PE, and IL2Rß-FITC, were purchased from PharMingen (San Diego, CA). The hybridoma, KJ1-26, which reacts to the clonotype TCR of DO.11.10 mice, was the kind gift of Dr. Philippa Marrack (National Jewish Center for Immunology and Respiratory Diseases, Denver, CO). KJ1-26 and mAb to mouse TCR-ß, clone H57-597, was purified from mouse ascites on HiTRAP Protein G columns (Pharmacia Biotech, Piscataway, NJ) and then conjugated to fluorescein (Sigma, St. Louis, MO) using established methods (27). Fluorescein-conjugated Ab was purified from reaction components by chromatography on PD-10 columns (Pharmacia Biotech).

Single cell suspensions were made by homogenizing tissues in RPMI 1640 medium (Life Technologies) supplemented with 5% (v/v) bovine calf serum (HyClone Laboratories, Ogden, UT). Cells excluding trypan blue were counted. Purification of CD4^-8^- thymocytes was accomplished using hybridoma culture supernatants from IgM anti-CD4, RL172.4, and IgM anti-CD8, 3.155, and complement as previously described (5). For flow cytometry, 10⁶ cells were incubated in 0.1 ml PBS containing 0.5% BSA fraction V, 0.001% (w/v) sodium azide (Sigma), and the indicated Abs (3 µg/ml) at 4°C for 30 min. After washing with PBS-azide, cells were fixed in 1% (v/v) methanol-free formaldehyde (Ted Pella, Reading, CA) in PBS-azide. Samples were stored at 4°C until analysis with a Coulter Elite flow cytometer calibrated using DNA check beads (Coulter, Hialeah, FL). Data were gated by forward and side light scatter using Elite software Cytomed (Coulter). Negative controls were set by using isotype-matched Ig directly conjugated to fluorochromes (Caltag).

TUNEL assay for apoptosis

Cells were initially stained for expression of TCR-ß, CD4, and CD8 and then fixed for 15 min at 4°C in 1% formaldehyde. Cell membranes were then permeabilized for 15 min using 70% ethanol at 4°C. Samples were incubated at 37°C for 1 h in 50 µl containing 10 U terminal deoxyribosyltransferase and 0.5 nM dUTP-biotin (Boehringer Mannheim, Indianapolis, IN) (28). Specimens were washed twice with PBS/1% BSA and incubated with a 1:50 dilution of streptavidin-tricolor (Caltag) at 4°C for 30 min. Cells were washed twice and analyzed by flow cytometry.

OVA peptides and treatment of TCR transgenic mice

Peptides to chicken OVA_323–339 (ISQAVHAAHAEINEAGR) (OVA_II), OVA_257–264 (SIINFEKL) (OVA_I), or OVA_I variants E1 (EIINFEKL) and R4 (SIIRFEKL) were produced at Macromolecular Resources (Colorado State University, Fort Collins, CO). Control peptide consisted of the calcineurin phosphatase substrate DLDVPIPGRFDRRVSVAAE (Bachem Biosciences, King of Prussia, PA). OVA_II/I-A^d binds to the DO.11.10 TCR with an affinity of 31 µM (29). OVA_I/K^b binds to the OT-1 TCR with high affinity (K_d = 6.5 µM), whereas E1 and R4 bind with lesser affinities of 22.6 and 57.1 µM, respectively (30). Unless otherwise noted, mice received 250 µl injections i.p. of 100 µM peptide solutions in PBS or PBS alone. Tissues were harvested 20 h later.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Apoptotic thymocytes are enriched for expression of TCR-ß and absence of CD4 and CD8

To define the subset of normal thymocytes undergoing apoptosis, the TUNEL assay was combined with Ab staining for surface expression of CD4, CD8, and TCR-ß. Fig. 1 illustrates that 7.4% of freshly isolated normal thymocytes, without further in vitro incubation, contained nicked DNA. Compared with the nonapoptotic thymocytes (Fig. 1, top row), the apoptotic subset (Fig. 1, bottom row) was markedly enriched for a CD4^-8^- phenotype (5% vs 21%). Equally striking were the differences in the surface levels of TCR-ß expression in apoptotic vs nonapoptotic thymocytes within the subsets of CD4⁺8⁺ and CD4^-8^- cells. Among nonapoptotic thymocytes, surface TCR-ß was quite low on the CD4⁺8⁺ subset (10.1%) and almost negligible on CD4^-8^- cells (2.8%). By contrast, the apoptotic cells in both subsets were considerably enriched for intermediate levels of TCR-ß expression, being 64.9% on CD4⁺8⁺ cells and 52.4% on CD4^-8^- thymocytes (Fig. 1). These findings were very consistent in three separate experiments. It should be noted that the intermediate levels of TCR-ß expressed by the apoptotic CD4^-8^- thymocytes is very similar to the TCR surface levels observed for both NK1⁺ T cells and lpr CD4^-8^- T cells (6). The enrichment for TCR-ß expression among apoptotic thymocytes is consistent with thymic negative selection occurring on cells that bear surface TCR.

View larger version (31K): [in this window] [in a new window]   FIGURE 1. Thymocytes undergoing apoptosis are enriched in TCR-ß+ cells within the CD4-8- and CD4+8+ subsets. Thymocytes from MRL+/+ mice were stained directly after isolation for expression of TCR-ß, CD4, and CD8, fixed, and then stained for nicked DNA using the TUNEL assay. Note the enrichment in the apoptotic subset (lower panel) for CD4-8- cells (21%). Furthermore, there is considerable enrichment for expression of TCR-ß in the subset undergoing apoptosis. Number inserts indicate percent positive cells. Positive control for TUNEL staining in the small histogram at left used thymocytes from an MRL+/+ mouse that had received 1 mg dexamethosone i.p. 18 h previously. The negative control eliminated only the dUTP-biotin step from the TUNEL protocol.

Up-regulation of CD5, CD69, and TCR/CD3 have been noted with positive selection, whereas down-regulation of heat stable Ag (HSA) and CD45RB have been observed with apoptosing thymocytes (19). Nonetheless, there was considerable overlap of the surface levels of HSA and CD45RB between the apoptotic and nonapoptotic subsets (19) As such, no single surface marker was observed to be restricted to the apoptotic subset. We elected to examine two additional markers based on parallels with lpr CD4^-8^- T cells as well as reports in other systems which suggested that they might serve as useful markers of apoptosis. B220, the B cell isoform of CD45, is expressed by the unusual TCR-ß⁺ CD4^-8^- cells that accumulate in lpr mice but by few T cells in normal mice (2). We have previously hypothesized that the TCR-ß⁺ CD4^-8^- B220⁺ phenotype arises from high intensity TCR signals that lead to apoptosis in normal mice, whereas this population is retained in lpr mice (23). The second marker was IL2Rß. Although the normal thymus expresses very little IL2Rß, it was highly expressed on thymocytes from a K^b-reactive TCR transgenic mouse that had been bred onto a negative selecting C57BL/6 background (21). The thymocytes in these mice were also almost exclusively devoid of surface CD4 and CD8. IL2Rß is also expressed by NK1⁺ T cells (5, 6).

Fig. 2A shows that staining of normal thymocytes for the activation markers CD44 and CD69 revealed that only a minor portion of the total thymocytes that expressed these markers actually contained nicked DNA, consistent with a previous report (19). Furthermore, among the apoptotic thymocytes, CD44 and CD69 expression was bimodal, containing significant proportions of CD44^- and CD69^- cells. Most of the CD44⁺ and CD69⁺ thymocytes were in fact not undergoing apoptosis. In contrast, IL2Rß and B220 were expressed by a smaller portion of thymocytes and these were considerably enriched for apoptotic cells. As many as two-thirds of the IL2Rß⁺ thymocytes and one-third of the B220⁺ subset contained nicked DNA (Fig. 2A). The IL2Rß and B220 subsets were extensively overlapping, suggesting that these two markers may be jointly expressed on many apoptosing thymocytes (Fig. 2B). However, some B220⁺ thymocytes lacked IL2Rß expression and vice versa. When total thymocytes were gated on the B220⁺ IL2Rß⁺ cells and further analyzed for expression of CD4 or CD8, 46.7% lacked expression of surface CD4 or CD8 and the remainder of this subset had only low levels of CD4 and CD8, in contrast to the high levels expressed by 82% of total thymocytes (Fig. 2B). This further supports the notion that apoptotic thymocytes are enriched in the CD4^-8^- subset.

View larger version (36K): [in this window] [in a new window]   FIGURE 2. Apoptotic thymocytes express IL2Rß and B220. A, Freshly isolated thymocytes were stained for expression of either TCR-ß, CD69, CD44, IL2Rß, or B220, and then analyzed for nicked DNA by TUNEL assay. Note that the majority of CD69+ or CD44+ thymocytes lacked nicked DNA. By contrast, the majority of IL2Rß+ thymocytes, and about one-third of B220+ thymocytes contained nicked DNA. B, B220 and IL2Rß identify a largely overlapping subpopulation of thymocytes which expresses either low or negligible (46.7%) levels of surface CD4 and CD8 compared with total thymocytes.

We have previously observed that the absence of CD2 on T cells from either lymph nodes (LN) in lpr mice (31), or intestinal intraepithelial lymphocytes (IEL) in normal mice (32), correlates with unresponsiveness to a variety of stimuli. As both lpr T cells and IEL undergo rapid apoptosis ex vivo (33, 34), we considered the possibility that the down-modulation of CD2 might also be a feature of T cells undergoing apoptosis. Consistent with this view is that while CD2 was highly expressed on most thymocytes, it was absent from a subset of TCR-ß⁺ CD4^-8^- thymocytes undergoing the most active apoptosis. As shown in Fig. 3, about one-third of the cells in this subset lacked CD2 expression. The percentage varied between 20 and 35% among different normal mouse strains (J.Q.R., unpublished observations). CD4^-8^- thymocytes were prepared and examined after incubation at 37°C for either 7 h or 18 h, for expression of TCR-ß and CD2 as well as nicked DNA. After 7 h the TCR-ß⁺ CD4^-8^- subset that expressed CD2 showed modest amounts of nicked DNA (27%) (Fig. 3). By contrast, the CD2^- subset manifested a markedly increased proportion of apoptotic cells (41%). By 18 h the proportion of apoptosis in the CD2^- subset (63%) increased substantially, whereas the CD2⁺ still manifested considerably less apoptosis (33%). This finding was consistent in two other experiments.

View larger version (24K): [in this window] [in a new window]   FIGURE 3. The absence of CD2 identifies a minor subset of TCR-ß+ CD4-8- thymocytes that is undergoing more rapid apoptosis. CD4-8- thymocytes from MRL+/+ mice were prepared by Ab and complement depletion and then analyzed after 7 h or 18 h at 37°C for TCR-ß+, CD2, and nicked DNA. Note that at both time points the subset lacking CD2 expression contained a higher proportion of nicked DNA than the CD2+ subset. Numbers in parentheses indicate the proportions of CD2- (left quadrants) or CD2+ (right quadrants) cells with nicked DNA.

The TCR-ß⁺ CD4^-8^- subset of LN cells contains self-reactive T cells that express B220

Collectively, the above findings suggested that the down-modulation of CD4, CD8, and CD2, concomitant with an increase in B220 and IL2Rß expression, identifies an apoptotic subset of normal thymocytes. This bears close phenotypic similarity to the phenotype of the TCR-ß⁺ CD2^- CD4^-8^- B220⁺ cells that accumulate in lpr mice. This model was studied further by examining the phenotypes of LN T cells bearing various TCR-Vß that either were or were not self-reactive based on the expression of endogenous mouse MTV. C57BL/6 mice bear MTV 8 and 9, but lack expression of class II MHC I-E and consequently are known to not efficiently delete MTV 8/9-reactive Vß5⁺ T cells (35). Consequently, these mice served as controls for levels of Vß and B220 expression. MRL^+/+ mice contain endogenous MTV 8 and 9 that lead to deletion of TCR-Vß5, whereas BALB/c mice express MTV 6, 8, and 9 that delete both Vß3 and Vß5 (35). As shown in Fig. 4A, C57BL/6 mice contained only a modest proportion of B220⁺ T cells among any of the three Vß examined. Similarly, as Vß8 T cells are not deleted in any of the three mouse strains, Vß8⁺ cells from all three mouse strains manifested little B220 expression. In contrast, Vß3⁺ cells in only BALB/c mice, and Vß5⁺ cells in both MRL^+/+ and BALB/c mice contained high proportions of B220⁺ cells among the minor amounts of these T cells that were present. These findings were based on 10⁵ accumulated events by FACS in each of the two highly consistent experiments, as summarized in Table I.

View larger version (32K): [in this window] [in a new window]   FIGURE 4. Self-reactive T cells, based on MTV expression, are largely contained in the B220+ subset, most of which lack CD4 and CD8. A, LN cells were analyzed from C57BL/6 mice (MTV 8+, 9+) which do not efficiently delete MTV-reactive T cells, or from MRL+/+ mice (MTV 8+, 9+) which delete Vß5+ T cells, or from BALB/c mice (MTV 6+, 8+, 9+) which delete Vß3+ and Vß5+ T cells. LN cells were stained for expression of B220 and the indicated Vß. Numbers in parentheses indicate the proportion of B220+ cells within the fraction expressing a given Vß. Note that B220 expression is prominent on Vß3+ cells only in BALB/c mice, on Vß5+ cells from both BALB/c and MRL+/+ mice, and not for Vß8 in any of the three mouse strains. B, BALB/c LN cells were stained with Abs to B220, CD4, CD8, and the indicated Vß. Note that both Vß3+ and Vß5+ cells are most prevalent in the CD4-8- subset and the majority express B220. The opposite is true for the Vß8+ subset. C, The same profiles as in B but gated to show the CD4 vs CD8 distribution on the B220- and B220+ subsets of the indicated Vß.

Together these findings correlate the phenotype, TCR-ß⁺ CD4^-8^- B220⁺, of peripheral T cells with reactivity to endogenous superantigens. To determine whether the actual affinity of the TCR interaction with peptide/MHC was the critical signal that provoked the CD4^-8^- B220⁺ phenotype in responding T cells, two different TCR transgenic mice were used, one restricted to class I MHC and the other to class II MHC. This provided the ability to manipulate the TCR signal intensity by altering either the dose of Ag or the affinity of peptide variants for the TCR.

In vivo induction of the phenotype, TCR-ß⁺ CD2^- CD4^-8^- B220⁺, results from a high intensity TCR signal that reflects its affinity for Ag/MHC

The two TCR transgenic mice used each recognize chicken OVA, the first being restricted to class I MHC (OT-1) and the other to class II MHC (DO.11.10). The OT-1 mouse recognizes an 8-aa OVA peptide, SIINFEKL (OVA_I), restricted to K^b (26). The affinity of the OVA_I/K^b complex for the transgenic OT-1 TCR has been defined using plasmon resonance for the native OVA_I peptide, and several single amino acid substitutions. OVA_I has an affinity of 6.5 µM, whereas E1 (EIINFEKL) has a K_d of 22.6 µM and R4 (SIIRFEKL) a K_d of 57.1 µM (30). These differences in affinity result in very different effects on the phenotype of the V2⁺ transgenic thymocytes in fetal thymic organ cultures, as well as function based on K^b stabilization, CTL responses, and proliferation (Refs. 26 and 36, and our unpublished results). The affinity of the DO.11.10 TCR for OVA_II/I-A^d has been measured at 31 µM (29).

To compare the effects of identical amounts of peptide delivered to the class I vs class II OVA systems, a standard dose of 250 µl of 100 µM peptide was administered i.p. to the TCR transgenic mice, and after 20 h the thymus and LN were examined. This dose was established in preliminary studies as it produced an intermediate level of reduction of thymocyte number and an increase in LN cell number at 20 h in both transgenic mice.

Administration of OVA_I, E1, or R4 peptides to OT-1 mice produced decreases in the cell number of all thymocyte subsets. Some of this effect may have resulted from the secondary stress of widespread T cell activation in the thymus. Nonetheless, only high affinity OVA_I produced a slightly increased proportion (though not absolute number) of V2⁺ CD4^-8^- B220⁺ thymocytes (data not shown).

In the LN of OT-1 mice the effects of the OVA peptides on cell number and phenotype were more dramatic. This is shown for one experiment in Fig. 5 and summarized for all three studies in Table II. Neither control peptide nor R4 significantly altered the phenotype of LN cells compared with PBS. E1 yielded a slight though inconsistent increase in LN cell number whereas OVA_I produced a 2- to 3-fold increase. Furthermore, none of the peptides significantly altered the number of CD8⁺ cells, although OVA_I consistently yielded a dramatically decreased percentage of CD8⁺ cells in all three experiments, and a decrease in absolute number in two of the three experiments (Table II). As shown for experiment 1 in Fig. 5, following OVA_I, the proportion of CD8⁺ cells decreased from 60% in control mice to 20% with OVA_I. In addition, the residual CD8⁺ cells after OVA_I contained a higher percentage of B220⁺ cells (23% compared with 11% or 9% with PBS or control peptide, respectively). Concomitant with the decrease in CD8⁺ cells, OVA_I consistently produced a markedly increased proportion of CD4^-8^- cells, resulting in an increased absolute number of V2⁺ CD4^-8^- cells. This was statistically significant in each of the three experiments (Table II). Although the B cells (B220⁺ V2^- in the CD4^-8^- subset) decreased by proportion following OVA_I, their absolute numbers increased due to the extensive expansion of LN cell numbers. Again, this may have resulted from secondary effects of widespread T cell activation.

View larger version (82K): [in this window] [in a new window]   FIGURE 5. Induction of V2+ CD4-8- B220+ LN cells in OT-1 mice receiving high affinity OVAI peptide. OT-1 mice received 250 µl containing either PBS or a 100 µM solution of control peptide, low affinity OVA peptides R4 (SIIRFEKL, Kd = 57.1 µM), E1 (EIINFEKL, Kd = 22.6 µM), or high affinity OVAI (SIINFEKL, Kd = 6.5 µM). After 20 h LN cells were enumerated and phenotyped. Note the increased absolute number of V2+ CD4-8- cells with high affinity OVAI, and to a lesser extent with lower affinity E1, but not with the lowest affinity R4 or control peptide. The increase was statistically significant (*) for OVAI in each of three experiments, but for E1 in only one of three studies (see Table II). In addition, OVAI administration resulted in an increased proportion of B220+ LN cells within the CD8+ cells. Numbers in parentheses indicate the proportion of V2+ cells that are B220+.

View this table: [in this window] [in a new window]   Table II. High affinity OVAI peptide induces increase of V2+ CD4-8- OT-1 LN cells1

Induction of IL2Rß and apoptosis was also observed in LN T cells with high affinity OVA_I. Fig. 6A illustrates that, similar to B220, IL2Rß was already expressed by more than 60% of the V2⁺ CD4^-8^- cells even in PBS control OT-1 mice. However, little IL2Rß was observed on the V2⁺ cells in the CD4⁺ and CD8⁺ subsets after PBS (10%), but this proportion increased considerably to 70% 20 h after OVA_I administration. As with the thymus, expression of IL2Rß and B220 occurred on partly overlapping though not entirely coincident subpopulations In addition, evidence of apoptosis by the TUNEL assay was most apparent in the CD4^-8^- subset of T cells following OVA_I (Fig. 6B). Whereas the V2⁺ cells within the CD4⁺ and CD8⁺ subsets contained very few cells bearing nicked DNA 20 h after OVA_I treatment, the CD4^-8^- subset of V2⁺ cells was highly enriched for apoptotic cells. As in the thymus, the apoptotic subset expressed low levels of surface TCR compared with the viable T cells. Furthermore, if the V2⁺ CD4^-8^- cells were further subdivided based on B220 expression, apoptotic cells were observed in both subsets, but a much higher proportion of the the B220⁺ V2⁺ cells contained nicked DNA (64%) compared with the B220^- V2⁺ cells (39%). Very little apoptosis was observed within the population of B cells (B220⁺ V2^-). These findings are consistent with the view that the CD4^-8^- subset of T cells are enriched for apoptosis following high affinity Ag, as this is particularly manifest within those T cells expressing B220.

View larger version (48K): [in this window] [in a new window]   FIGURE 6. Induction of IL2Rß expression and apoptosis in V2+ cells following OVAI. LN cells from OT-1 mice were analyzed 20 h after OVAI administration and stained for expression of CD4, CD8, V2, B220, and either (A) IL2Rß or (B) nicked DNA by TUNEL assay. Number inserts indicate the percentage of total cells in the indicated quadrants. Numbers in parentheses in B indicate the proportion of V2+ cells with nicked DNA.

View larger version (39K): [in this window] [in a new window]   FIGURE 7. Down-modulation of CD2 with high affinity OVAI. Since V2 expression was low on the CD4-8- subset of splenocytes following peptide administration, Thy 1.2 staining was used to better distinguish the T and B cell subsets. Within the CD8+ subset, OVAI but not E1 produced a decreased expression of CD2. The Thy 1.2+ cells in the CD4-8- subset manifested a considerable loss of CD2 expression that was decreased with E1 and diminished even further with OVAI. Numbers in parentheses represent the percentage of Thy 1.2+ cells lacking CD2 expression.

View larger version (40K): [in this window] [in a new window]   FIGURE 8. Lack of induction of CD4-8- T cells or B220+ in DO.11.10 mice. Three DO.11.10 mice per group received either a single administration of PBS or OVAII, or three 250 µl injections of 100 µM OVAII at 24-h intervals. LN cells were analyzed 20 h after the last injection. Even with multiple doses of OVAII, despite a marked increase in LN cell number, there was no increase of CD4-8- KJ1-26+ cells or induction of B220.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

When our results are combined with those of Kishimoto et al. (19), a model can be developed which proposes that a cascade of changing phenotypes occurs on developing thymocytes or mature peripheral T cells that reflects the intensity of the TCR/costimulatory signals. At the lower intensity is the null TCR interaction, which results in deletion by neglect. At the upper extreme is the highest intensity TCR signal which provokes deletion and down-regulation of CD2, CD4, and CD8 with concomitant expression of B220. In between these extremes is a broad range of TCR signal intensities that allows cell survival.

The model may serve to explain the phenotype of thymocytes from three TCR transgenic mice when bred onto self-reactive backgrounds that presumably confer a high intensity TCR signal. These are the H-Y-specific (20), K^b-specific (21), and DO.11.10 (22) TCR transgenic mice. In the case of the H-Y-specific TCR transgenic mice, female mice (lacking H-Y) positively select the TCR transgene on CD8⁺ thymocytes. By contrast, male (H-Y⁺) transgenic mice manifest a small thymus comprised of predominantly TCR-ß⁺ CD4^-8^- thymocytes (20). Similarly, in H-2K^b-reactive TCR transgenic mice, expression of the TCR transgene in nonselecting H-2^d mice (null TCR signal) yields few mature thymocytes, as development is arrested at the CD4⁺8⁺ stage (21). Expression in H-2^k mice results in positive selection of CD8⁺ thymocytes lacking IL2Rß (moderate TCR signal). Finally, expression of the K^b-reactive transgenic TCR in K^b mice produces almost exclusively CD4^-8^- IL2Rß⁺ thymocytes bearing the clonotype TCR transgene (high intensity TCR signal) (21). A third example occurs in the same DO.11.10 TCR transgenic mouse used in this study. Although the response to OVA_II is restricted to I-A^d, the TCR also bears a cross-reactive alloresponse to H-2^b. When bred onto the H-2^b background, DO.11.10 mice manifest primarily CD4^-8^- thymocytes and LN cells bearing the TCR transgene (22). It is not certain in these specialized transgenic situations where the TCR may be prematurely expressed at the CD4^-8^- stage, whether the CD4^-8^- T cells were derived from precursors that previously expressed CD4 and/or CD8. This result has been reported only in the DO.11.10/H-2^b mice where the CD4^-8^- T cells contained a partially demethylated CD8 gene, consistent with at least some previous expression of CD8 (22). However, our current model would allow that in TCR transgenic mice bearing early expression of a self-reactive TCR, initial up-regulation of surface CD4 or CD8 could be prematurely blocked. Yet the resulting CD4^-8^- phenotype would still be due to the same physiology as in nontransgenic T cells that received a high intensity TCR signal from a self-Ag.

Certain other predictions from this model have been observed. First is that if a self-reactive TCR signal manifests as a high intensity signal in the thymus or periphery, then the TCR-ß⁺ CD4^-8^- subset should be enriched for self-reactive T cells. Indeed, we observed this to be the case for LN T cells, based on the expression of mtv-encoded superantigens. Furthermore, a high proportion of these self-reactive CD4^-8^- T cells expressed B220. In this regard, it is of interest that an increase in TCR-ß⁺ CD4^-8^- cells has been reported in patients with active systemic lupus erythematosus (24).

A second prediction is that manipulations that diminish the intensity of TCR signaling would shift the resulting phenotype away from CD4^-8^- toward CD4⁺ and CD8⁺ T cells. Urdahl et al. (37) made such observations in the thymus using cyclosporin A to functionally diminish TCR signal intensity. Administration of cyclosporin A to male H-Y-specific TCR transgenic mice resulted paradoxically in a larger thymus with fewer CD4^-8^- and more CD8⁺ thymocytes.

An unresolved issue from these studies is whether there is a link between the CD4^-8^- T cells observed in these studies or in lpr mice, and murine NK1⁺ CD4^-8^- T cells that express an invariant TCR -chain, V14-J281, react to CD1d, and produce large quantities of IL-4 upon activation (4, 5, 6, 7). Beyond their CD4^-8^- phenotype, NK1⁺ T cells appear at first analysis to have little in common with the other CD4^-8^- T cells. Almost certainly their lineage is different in terms of what Ag specificity gives rise to them. However, the physiology of their origin may not be so different, as it relates to intensity of TCR signaling. In this regard, an earlier study by Erard et al. (38) may provide the best clue to their similar origins. These investigators demonstrated that purified peripheral CD8⁺ cells from normal mice became CD4^-8^- when activated with the combination of PMA, ionomycin, IL-2, and IL-4. IL-4 was critical to this process and CD4⁺ cells did not undergo this conversion. Furthermore, the transition from CD8⁺ to CD4^-8^- in vitro over 3 to 4 days was permanent, and the resulting CD4^-8^- T cells were now high producers of IL-4. Conceivably, the presence of IL-4 during the delivery of a high intensity TCR signal might rescue the cells from apoptosis and preserve their CD4^-8^- phenotype while promoting a Th2 cytokine pattern upon them. In preliminary studies we have reproduced the findings of Erard et al. (38) using OVA_I and IL-4 in vitro to induce OT-1 CD8⁺ cells to become CD4^-8^- (J.Q.R., unpublished observations). Thus, NK1⁺ T cells and other CD4^-8^- T cells may have a similar physiology of origin, even if their Ag specificities and TCR repertoire differ. A prediction from our model would thus be that the TCR of NK1⁺ T cells would have a high affinity interaction with CD1d.

Two additional points from this study bear note. The first is that the TCR-ß⁺ CD4^-8^- B220⁺ phenotype was much more efficiently induced in the class I-restricted OT-1 mice than in the class II-restricted DO.11.10 mice. This may merely reflect a higher affinity by chance of the OVA_I/K^b complex for OT-1 TCR (6.5 µM) than OVA_II/I-A^d for DO.11.10 TCR (31 µM). However, the added fact that in the absence of class I MHC in normal or lpr mice, most TCR-ß⁺ CD4^-8^- cells are absent (11, 12, 13, 14), is consistent with a view that on average, signaling via class I confers a higher intensity signal to CD8⁺ T cells than via many class II-restricted peptides to CD4⁺ T cells. This might also explain the slightly greater frequency of B220 expression on CD8⁺ than CD4⁺ cells in normal mice (Fig. 4B). This view is also consistent with a model from Goldrath et al. (36), suggesting that during thymic selection CD8 lineage cells are normally selected at higher TCR-peptide/MHC affinities than are CD4⁺ cells.

The second point is that while the dose of peptide in our system affected cell numbers of thymocytes and LN, it was the peptide affinity that most closely determined the outcome phenotype. This notion has also been appreciated in fetal thymic organ cultures of OT-1 mice using the same OVA peptides as in the current study (26). TCR-peptide/MHC affinities vary more in their off rates (k_off) rather than their on rates, and k_off is independent of Ag concentration (30). This suggests that k_off and hence duration of TCR occupancy may be a pivotal factor in the intensity of TCR signaling and phenotypic outcome.

In summary, the current findings support a model where the continuum of TCR signal intensities parallels particular phenotypic outcomes and cell fates. While the general principle of the model may be applicable to both thymocytes and mature peripheral T cells, the outcome phenotype and cell fate in response to a given TCR signal may be somewhat different at these two sites. The same signal that delivers an intermediate signal intensity and proliferation to mature T cells may cause profound deletion of thymocytes. Thus, although the absolute signal intensity that confers a given phenotype may vary between developing and mature T cells, the physiology is nonetheless similar, with high intensity TCR signaling leading to down-regulation of CD2, CD4, and CD8, and up-regulation of B220 and IL2Rß. This phenotype may therefore identify potentially autoreactive T cells that are increased in certain autoimmune syndromes (24).

	Acknowledgments

 
We thank Drs. Mercedes Rincon and Karen Newell for helpful discussions with the manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant AI36333 (to R.C.B.) and AR08267 (to P.F.M.).

² Current address: Department of Microbiology, Washington State University, Pullman, WA 99164-4233.

³ Address correspondence and reprint requests to Dr. Ralph C. Budd, Immunobiology Program, University of Vermont College of Medicine, Given Medical Building, C-303, Burlington, VT 05405-0068.

⁴ Abbreviations used in this paper: CD4^-8^-, CD4^-CD8^-; MTV, mammary tumor virus; OVA_II, OVA peptide 323–339; OVA_I, OVA peptide 257–264; LN, lymph nodes.

Received for publication June 12, 1998. Accepted for publication February 19, 1999.

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