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Chronic Modulation of the TCR Repertoire in the Lymphoid Periphery¹

Department of Immunology, School of Medicine, University of Washington, Seattle, WA 98195

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Using TCR Vß5 transgenic mice as a model system, we demonstrate that the induction of peripheral tolerance can mold the TCR repertoire throughout adult life. In these mice, three distinct populations of peripheral T cells are affected by chronic selective events in the lymphoid periphery. First, CD4⁺Vß5⁺ T cells are deleted in the lymphoid periphery by superantigens encoded by mouse mammary tumor viruses-8 and -9 in an MHC class II-dependent manner. Second, mature CD8⁺Vß5⁺ T cells transit through a CD8^lowVß5^low deletional intermediate during tolerance induction by a process that depends upon neither mouse mammary tumor virus-encoded superantigens nor MHC class II expression. Third, a population of CD4^-CD8^-Vß5⁺ T cells arises in the lymphoid periphery in an age-dependent manner. We analyzed the TCR V repertoire of each of these cellular compartments in both Vß5 transgenic and nontransgenic C57BL/6 mice as a function of age. This analysis revealed age-related changes in the expression of V families among different cellular compartments, highlighting the dynamic state of the peripheral immune repertoire. Our work indicates that the chronic processes maintaining peripheral T cell tolerance can dramatically shape the available TCR repertoire.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Throughout life, the immune system must maintain a balance between the need to respond to the universe of foreign Ags and the need to maintain tolerance to self-Ags. Among T cells, this balance is maintained by both thymic and peripheral selective events. In the thymus, developing thymocytes are subject to both positive and negative selection, processes which together tailor the population of mature self-tolerant T cells to recognize foreign peptides in the context of self-MHC molecules 1, 2 . Once mature T cells have left the thymus, a variety of peripheral events continue to modulate the T cell repertoire. For example, viral infection can cause a dramatic expansion of Ag-specific T cells, resulting in over-representation of a few TCR specificities 3, 4 . Tolerance induction in the lymphoid periphery can also shape the T cell repertoire through a variety of mechanisms, including clonal deletion 5, 6 , clonal diversion 7, 8 , clonal exhaustion 9, 10 , and clonal anergy with or without an associated down-regulation of the TCR and/or accessory molecule expression on autoaggressive cells 11, 12, 13, 14, 15, 16, 17, 18 .

Our studies of the induction of peripheral tolerance use as a model system C57BL/6 (B6)³ mice (H-2^b, I-E^-) transgenic (Tg) for a rearranged TCR Vß5.2 chain 17, 19, 20 . An advantage of this strain is that Vß5⁺ T cells can be readily followed in vivo during responses to superantigens (SAgs), which interact with T cells largely through the TCR Vß-chain (for review see 21 . In addition, the limited diversity of the TCR repertoire in Vß5 Tg mice permits us to study perturbations in the immune repertoire within a relatively homogenous population of cells. Previous studies of tolerance induction among Vß5⁺ T cells have demonstrated that Vß5⁺ T cells in MHC class II I-E⁺ mice are deleted intrathymically by vSAG9, a SAg encoded by the endogenous mouse mammary tumor virus (Mtv)-9 19, 22, 23, 24, 25 . In MHC class II I-E^- Vß5 Tg mice, mature CD4⁺ and CD8⁺ T cells escape the thymus, but are selected against in the lymphoid periphery by endogenous self-Ags 17, 19, 20 . Peripheral CD4⁺Vß5⁺ T cells are activated and rendered anergic before their deletion 20 , while the chronic and incomplete deletion of peripheral CD8⁺Vß5⁺ T cells correlates with the formation of CD8^lowVß5^low cells, defined as deletional intermediates 17 . We have now evaluated how the distinct tolerance pathways taken by these cells influence the TCR V repertoire. While the thymus is responsible for molding the preimmune TCR repertoire, age-related changes in TCR V expression among different cellular compartments in both Vß5 Tg and non-Tg B6 mice emphasize that postthymic events can also modify the TCR repertoire. Our studies show that, even after thymic involution, poorly expressed, weak tolerogens in the lymphoid periphery can induce dramatic and long-term alterations in the immune repertoire.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

B6 Vß5 and B6.OT-1 TCR Tg mice were derived to express either the Vß5 chain only (Vß5 Tg), or the V2 and Vß5 chain (OT-1 Tg) from a CD8⁺ CTL clone specific for chicken OVA and H-2K^b and have been described previously 19, 26 . These mice carry the endogenous proviral integrants Mtv-8, -9, -17, and -30. Tg mice were maintained as heterozygotes by crossing Vß5 Tg or B6.OT-1 Tg mice with B6 mice purchased from The Jackson Laboratory (Bar Harbor, ME) and by screening for Vß5 expression by flow cytometry. Non-Tg mice were offspring from the same matings. Vß5 Tg B6 x BXD15 mice (Mtv-1, -6, -8, -9, -11, -13, -17, and -30) were obtained by crossing Vß5 Tg B6 mice to the H-2^b, I-E^- BXD15 recombinant inbred line (The Jackson Laboratory). An Mtv^- line of Vß5 Tg mice and Vß5 Tg mice carrying single Mtv-integrants were derived by intercross/backcross breeding of B6 Vß5 Tg mice with a male WLC-0 mouse generously provided by Dr. D. Morris (University of California at Irvine, CA). WLC-0 mice were originally wild-derived and are I-E^- and Mtv^- by stringent molecular analyses 27, 28, 29 . Tg offspring were screened for the presence of Mtvs by Southern blot using a probe that cross-hybridizes with all endogenous Mtvs, as described 20 . Vß5 Tg MHC class II^-/- mice were obtained by crossing the Vß5 transgene onto MHC class II^-/- mice 30 purchased from The Jackson Laboratory and backcrossed to the B6 background through the 12th generation. Offspring were screened for the Vß5 transgene and the absence of both I-A and peripheral CD4⁺ T cells. B6.PL-Thy-1^a/Cy mice (referred to as B6.Thy1.1 mice; The Jackson Laboratory) were bred and maintained in our animal facility. Chimeric mice were generated by lethally irradiating (925 rad) B6.Thy1.1 mice and injecting 8–10 x 10⁶ T cell-depleted bone marrow cells from Vß5 Tg MHC class II^-/- mice (Class II^-/- wild-type (WT)) or Vß5 Tg B6 mice (WT WT), as previously described 31 . All animals were maintained in a specific pathogen-free barrier animal facility at the University of Washington.

Reagents

Phycoerythrin (PE)-conjugated-anti-CD8 (53-6.7) and -anti-CD4 (RM-4-5) mAbs, biotin-conjugated-anti-Thy-1.2 (30-H12) and -anti-V2 mAbs, and FITC-conjugated-anti-Vß5 (MR9-4) and -anti-Thy-1.2 (30-H12) mAbs were purchased from PharMingen (San Diego, CA). Unconjugated anti-CD8 (3168.8), anti-CD4 (RL172.4R6), and anti-Vß5 (MR9-4) Abs were obtained from ascites or tissue culture supernatants. Purified goat anti-mouse Ig and TriColor (TC)-conjugated streptavidin were purchased from Caltag Laboratories (South San Francisco, CA). Guinea pig complement and PCR primers were purchased from Life Technologies (Grand Island, NY).

Flow cytometry

Unless otherwise noted, lymph node (LN) cells were isolated from pooled inguinal, axial, brachial, cervical, and mesenteric LNs. PBL were obtained by water lysis of whole heparinized blood. Cells were stained as described previously 19 and analyzed on a FACScan using CellQuest software (Becton Dickinson, San Jose, CA). Unless otherwise noted, dead cells were excluded on the basis of forward and side scatter profiles, and a minimum of 10⁴ live gated events were collected. Cell sorting was performed on a FACStar^Plus with LYSYS II software (Becton Dickinson).

Purification of cell populations

CD4⁺ and CD8⁺ T cells from young (8–10 wk), middle aged (30 wk), and old (60–65 wk) B6 Vß5 Tg and non-Tg mice were enriched from pooled spleen and LN cells by Ab plus complement-mediated depletion of CD8⁺ and CD4⁺ T cells, respectively. Tg populations were stimulated for 4 days in anti-Vß5-coated flasks in the presence of 100 U/ml rIL-2 (Perkin-Elmer Cetus Corporation, Emeryville, CA), and transferred to uncoated flasks for 2 days to allow dissociation of bound anti-Vß5 mAb. Non-Tg cells were stimulated with ConA at 3 µg/ml (Calbiochem, San Diego, CA) for 3 days. CD4⁺ and CD8⁺ T cell blasts were then positively selected by panning on anti-CD4- or anti-CD8-coated plates. The purity of the populations was assessed by flow cytometry using non-cross-blocking Abs. Purification of CD4^-CD8^- (double negative (DN)) T cells from middle aged and old B6 Vß5 Tg mice was performed as for Tg CD4⁺ or CD8⁺ T cells, except that after anti-Vß5 stimulation, cells were panned on plates coated with both anti-CD4 and anti-CD8 mAbs. The nonadherent cells (DN) were removed and their purity assessed by flow cytometry. CD8^low and CD8^high cells were purified by flow cytometric sorting using nylon wool nonadherent splenocytes from 15- to 18-wk-old Vß5 Tg B6 x BXD15 mice.

RT-PCR

Total RNA was extracted from purified cell populations with guanidinium thiocyanate/phenol 32 and reverse transcribed to cDNA with avian myeloblastosis virus reverse transcriptase (Life Technologies) and random hexamer primers (Pharmacia, Piscataway, NJ). To quantitate cDNAs, threefold serial dilutions of the cDNA reactions were subjected to PCR using primers specific for the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) 33 for 30–35 cycles of 94°C, 1 min, 60°C, 1 min, and 72°C, 1 min on a DNA ThermalCycler 480 (Perkin-Elmer Cetus). Beginning with similar amounts of cDNA, threefold serial dilutions of cDNA were then subjected to PCR using a conserved C-specific primer paired with a V family-specific primer (Table I) for 30–35 cycles of 94°C, 1 min, 55°C or 60°C (as noted in Table I), 1 min, and 72°C, 1 min. Because the V4 primer cannot recognize V4.4, we designed a separate V4.4 primer. The specificity of the primer pairs was determined empirically by their ability to amplify cDNA from hybridomas expressing known V genes. PCR reaction products were electrophoresed on a 2% agarose gel, Southern blotted under alkaline conditions to zeta-Probe GT membrane (Bio-Rad, Hercules, CA), and detected with either an HPRT- or C-specific probe. Bands were quantitated on a Phosphorimager 425 using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). To normalize V expression levels to HPRT levels, the integrated volume of the V product was divided by the integrated volume of the HPRT product for each dilution. Because a different primer was used for each V family, no attempt was made to compare the frequencies of different V families. Instead, relative intensities of each V family between the cell subsets were analyzed.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Our previous studies of the induction of tolerance among mature peripheral T cells have characterized the chronic selection against both CD4⁺ and CD8⁺ T cells in TCR Vß5 Tg B6 mice 17, 19, 20 . Age-dependent deletion of CD4⁺ T cells and relatively stable numbers of CD8⁺ T cells in Vß5 Tg mice combine to drive the inversion of the CD4:CD8 ratio among peripheral T cells 19, 20 . This decline in CD4⁺Vß5⁺ T cells is also evident in non-Tg B6 mice, and is therefore not an idiosyncrasy of the transgene. Thus, in both Vß5 Tg and non-Tg B6 mice, CD4⁺Vß5⁺ PBL are deleted, leading to a decline in the CD4:CD8 ratio among Vß5⁺ T cells from 3:1 at 5 wk of age to <0.2:1 at 30 wk of age (Fig. 1). During this time frame, the CD4:CD8 ratio among total T cells in non-Tg B6 mice declines only slightly. This correlation between deletion and expression of a defined Vß element suggests that the tolerogen driving CD4⁺Vß5⁺ T cell deletion is a SAg.

View larger version (27K): [in this window] [in a new window]   FIGURE 1. The CD4:CD8 ratio inverts with age among Vß5+ T cells in both Tg and non-Tg mice. PBL from Tg and non-Tg mice aged 6–45 wk and spleen cells from mice aged 0–5 wk were stained with FITC-anti-Vß5 and PE-anti-CD4 or PE-anti-CD8 and analyzed by flow cytometry. The CD4:CD8 ratio and SEs among total T cells and Vß5+ T cells were calculated for 3–18 mice per age group.

To investigate which SAg drives the deletion of CD4⁺Vß5⁺ T cells, we generated lines of Mtv^- Tg and non-Tg mice (H-2^b, I-E^-) and strains carrying each of the Mtv genes found in B6 mice (Mtv-8, -9, -17, and -30) singly or in combination. At various ages, we examined PBL for evidence of CD4⁺Vß5⁺ T cell deletion, which is seen in the original B6 Vß5 Tg line as a dramatic decline in the CD4:CD8 ratio followed by a slight recovery after 1 year of age (Fig. 2A) and as a loss of Vß5 expression among peripheral CD4⁺ T cells (Fig. 2B). Unlike B6 Tg mice, Mtv^- Tg mice neither invert their CD4:CD8 ratio (Fig. 2A) nor lose Vß5 expression among peripheral CD4⁺ T cells (Fig. 2B), indicating that a vSAG is required for tolerance induction among CD4⁺Vß5⁺ T cells. Tg mice carrying either Mtv-17, Mtv-30, or both did not differ in their phenotype from Mtv^- Tg mice, indicating that these vSAGs do not drive deletion of CD4⁺Vß5⁺ T cells (data not shown). However, vSAG8 and vSAG9 can independently drive the deletion of CD4⁺Vß5⁺ T cells, because mice carrying Mtv-8, Mtv-9, or both recapitulate the original B6 Tg phenotype (Fig. 2).

View larger version (21K): [in this window] [in a new window]   FIGURE 2. Both Mtv-8 and Mtv-9 drive the deletion of CD4+ T cells in Vß5 Tg mice. A, Mtv-8 and Mtv-9 are responsible for the inversion of the CD4:CD8 ratio in Vß5 Tg mice. PBL from mice of the indicated backgrounds and ages were stained and analyzed as in Fig. 1 to determine the CD4:CD8 ratio. Each symbol represents data from an individual mouse. B, Both Mtv-8 and Mtv-9 can drive the loss of transgene expression in CD4+ peripheral T cells in Vß5 Tg mice. The percent Vß5+ T cells among CD4-gated cells was determined by flow cytometric analysis of PBL prepared as above; each symbol represents data from an individual mouse.

We explored the potential contribution of MHC class II I-A molecules to Vß5⁺ T cell tolerance by generating radiation chimeras in which bone marrow cells from Vß5 Tg MHC class II^+/+ (WT) or Vß5 Tg Class II^-/- mice were injected into irradiated B6.Thy1.1 mice. At each time point after reconstitution, Class II^-/- WT chimeras had significantly greater numbers of CD4⁺ T cells (Fig. 3, left, and data not shown) and a significantly greater CD4:CD8 ratio (Fig. 3, middle, and data not shown) than did WT WT chimeras. This indicates that the expression of MHC class II molecules on bone marrow-derived cells is required for CD4⁺ T cell deletion in Vß5 Tg mice.

View larger version (20K): [in this window] [in a new window]   FIGURE 3. MHC class II+ bone marrow-derived cells in Vß5 Tg mice are required for the deletion of CD4+ peripheral T cells but not for the formation of CD8low cells. Ten weeks after reconstitution, PBL from WT WT and Class II-/- WT radiation bone marrow chimeras were stained and analyzed by flow cytometry to determine the percent CD4+ T cells, the CD4:CD8 ratio, and the percent CD8low/total CD8+ T cells. Data represent results from four mice. Differences between WT WT and Class II-/- WT chimeras in the percent CD4 and the CD4:CD8 ratio were statistically significant (p < 0.001) by a Student’s t test. Differences in the percent CD8low cells were not statistically significant (p > 0.7). Similar results were obtained at 16, 21, and 26 wk postreconstitution.

In Vß5 Tg mice, CD8⁺ T cells transit through a well-defined CD8^lowVß5^low compartment during their deletion 17 . Although CD8^low cells are more frequent among Vß5⁺ cells, CD8^low cells can also develop among Vß5^- cells in non-Tg B6 mice (data not shown), indicating that the correlation between Vß5 expression and tolerance induction is not as tight among CD8⁺ T cells as among CD4⁺ T cells in B6 mice. The percentage of CD8^low cells in the PBL of Class II^-/- WT chimeras did not significantly differ from that in WT WT chimeras (Fig. 3, right), demonstrating that CD8^low formation does not require MHC class II expression on bone marrow-derived cells. In addition, CD8^low formation does not require vSAG expression, because CD8^low cells develop in both Mtv^- and Mtv-8⁺9⁺ Tg mice (Fig. 4A) and in Tg mice carrying other Mtvs (data not shown), though their formation may be slower in Mtv^- mice. These data demonstrate that the tolerogen triggering CD8^low formation differs from that driving CD4⁺ T cell deletion.

View larger version (32K): [in this window] [in a new window]   FIGURE 4. The formation of CD8low cells in Vß5 Tg mice is Mtv-independent but TCR-dependent. A, CD8low cells develop in Mtv- and Mtv-8+9+ Vß5 Tg mice, but not in B6 OT-1 Tg mice. The percent CD8low/total CD8+ T cells among PBL from Mtv- non-Tg, Mtv-8+9+ Tg, Mtv- Tg, and B6 OT-1 mice (3–5 mice per age group) was determined by analyzing CD8- and Vß5-stained cells by flow cytometry. B, The CD8low compartment in B6.OT-1 mice is enriched for cells that do not express the transgenic TCR . PBL from B6.OT-1 TCR Tg mice of the indicated ages were stained with FITC-anti-Vß5, PE-anti-CD8, and biotin-anti-V2 followed by TC-streptavidin. The percentage of V2- cells among CD8low and CD8high was determined by gating on CD8low or CD8high cells and analyzing the percent expressing V2. Results are from 3–7 mice per age group, and error bars represent SEs of the means.

The TCR V repertoire changes during aging and during the chronic induction of tolerance in Vß5 Tg and non-Tg B6 mice

To determine whether the TCR -chains expressed in Vß5 Tg mice play an important role in tolerance induction, we analyzed the peripheral V repertoire in both Tg and non-Tg B6 mice at three distinct ages: young (8–10 wk) before maximal CD4⁺ deletion or CD8^low formation has occurred, middle-aged (30 wk) at the peak of CD4⁺ T cell deletion, and old (60–65 wk) when CD4⁺ T cell numbers recover slightly. Despite very low frequencies of some cell subsets in middle-aged and old mice, we were able to isolate highly purified cell populations (Table II) by enriching for CD4⁺ and CD8⁺ T cells both before and after stimulation with either anti-Vß5 Abs (for cells from Tg animals) or ConA (for cells from non-Tg animals). We then compared the expression levels of V families between cell subsets (Fig. 5). The expression levels of V2, V4, V8, and V11 increase with age in both Tg and non-Tg animals (Fig. 5A), while V5, V7, V9/10, V13, and V18 expression levels are highest in young or middle-aged mice (Fig. 5). V1 is unusual in its poor expression among CD8⁺ T cells in both Tg and non-Tg animals (Fig. 5). Some V families (V6, V12, V14, V15, V16, V17, and V19) are poorly expressed in both Tg and non-Tg B6 mice and therefore were not quantitated. This analysis illustrates significant age-related changes in V expression in both Vß5 Tg and non-Tg B6 mice.

View larger version (57K): [in this window] [in a new window]   FIGURE 5. Expression of V family members changes in an age-related manner in both Vß5 Tg and non-Tg B6 mice. Relative expression levels of each V family, determined by RT-PCR as described in Materials and Methods, were normalized to HPRT and then to a maximum level of 10 within each V family and plotted for young, middle-aged, and old mice among Tg and non-Tg CD4+ and CD8+ T cells. Data are the average of 2–3 independent experiments, each using three cDNA dilutions from each subpopulation.

View this table: [in this window] [in a new window]   Table III. Ratio of HPRT-normalized V expression levels in Tg:non-Tg mice1

View this table: [in this window] [in a new window]   Table IV. Ratio of HPRT-normalized V expression levels in CD8low:CD8high cells1

CD4^-CD8^- (DN) Vß5⁺ T cells develop in aging Vß5 Tg and non-Tg mice and demonstrate selection for expression of a particular V-chain

A third compartment that alters with age in Vß5 Tg mice is the peripheral CD4^-CD8^- DN compartment. The percentage of DN peripheral T cells dramatically increases in an age-dependent manner among Vß5⁺ but not among Vß5^- T cells in Vß5 Tg mice (Fig. 6). In non-Tg B6 mice, the representation of DN cells is much greater among Vß5⁺ T cells than among Vß5^- T cells, and this over-representation is relatively stable in non-Tg mice of various ages (Fig. 6). To elucidate the origin and TCR repertoire of these coreceptor-null T cells, we compared the V repertoire of DN T cells to age-matched CD4⁺ and CD8⁺ T cells from Tg mice. DN T cells in middle-aged mice demonstrate increased expression of many V families relative to either CD4⁺ or CD8⁺ T cells (Table V). Poor expression of V1 among CD8⁺ T cells (Fig. 5) leads to an exaggerated expression of V1 among DN T cells relative to CD8⁺ T cells from old Tg mice. However, DN T cells in old mice demonstrate consistent over-representation of V4.4, and low expression of nearly all other V families (Table V). Therefore, the induction of peripheral tolerance in Vß5 Tg mice leads to the predominance of a subpopulation of DN T cells in old mice, many of which express a V4.4⁺Vß5⁺ TCR.

View larger version (23K): [in this window] [in a new window]   FIGURE 6. CD4-CD8- (DN) T cells are over-represented within the Vß5+ T cell compartment in both Vß5 Tg and non-Tg mice. PBL from Vß5 Tg and non-Tg mice of the indicated ages were analyzed by flow cytometry after staining with FITC-anti-Vß5 mAbs, biotin-anti-Thy-1.2 mAbs plus TC-streptavidin, and either PE-anti-CD4, PE-anti-CD8, or both mAbs. The percent DN of Vß5- PBL was determined directly by dividing the percent of Vß5-Thy-1.2+ cells that stained with neither anti-CD4 nor anti-CD8 mAbs by the percent of total Vß5-Thy-1.2+ cells to normalize to the total number of T cells. In Vß5 Tg mice, the percent Vß5- cells increases with age, due in part to rearrangement of endogenous TCR genes (63). The percent DN of Vß5+ T cells was calculated by adding the percent CD4+Vß5+ and the percent CD8+Vß5+ and subtracting this value from the percent Vß5+. This value was then divided by the percent Vß5+ to eliminate fluctuations due to age-related variation in the number of T cells. Error bars represent SEs of data averaged from 3–10 mice per age group. In Vß5 Tg mice, the percent DN T cells differed significantly (p < 0.05 by a paired Student’s t test) between Vß5+ T cells and Vß5- T cells in all age groups except 8–14 wk (when there are few Vß5- cells) and 43–60 wk. In non-Tg mice, the percent DN T cells differed significantly (p < 0.05 by a paired Student’s t test) between Vß5+ T cells and Vß5- T cells in each age range.

View this table: [in this window] [in a new window]   Table V. Ratio of HPRT-normalized V expression levels in CD4-CD8- DN T cells:CD4+ or CD8+ T cells1

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The CD8⁺ T cell compartment of Vß5 Tg mice is shaped by different factors than is the CD4⁺ T cell compartment in Vß5 Tg mice. MHC class II presentation of vSAGs does not drive the formation of CD8^low deletional intermediates, and the absence of a tight correlation between Vß5 expression and CD8^low formation makes it unclear whether a SAg (not one encoded by an Mtv) or a conventional Ag with a Vß preference drives CD8^low formation. While SAgs may be presented to T cells in the absence of MHC class II molecules and activate T cells bearing a specific ß-chain 43 , the CD8⁺ T cell responses of B6 mice to Moloney murine leukemia virus and OVA 44, 45 and the CD4⁺ T cell response of B10.A mice to pigeon cytochrome C 46 demonstrate a Vß preference. Therefore, either class of tolerogen could contribute to CD8^lowVß5^low formation and the modulation of the mature CD8⁺ T cell pool in Vß5 Tg mice.

An alternative tolerance pathway to CD4⁺ T cell deletion or CD8^low formation is coreceptor down-regulation to form DN T cells. This down-regulation may decrease the avidity of the interaction between a T cell and its tolerogen sufficiently to allow the T cell to escape deletion 14, 15 . The increased frequency of DN T cells among Vß5⁺ cells in non-Tg B6 mice (Fig. 6) in comparison to their normally minor representation among total T cells (Fig. 6 and Ref. 47–50) suggests that this "escape" pathway is common among Vß5^- T cells in B6 mice. The low frequency of DN T cells in Mtv^- mice (data not shown) suggests that DN T cells derive from CD4⁺Vß5⁺ T cells, not from peripheral CD8⁺ T cells or from distinct thymic precursor cells as has been suggested for the subpopulation of DN T cells characterized previously (for review see 51 . In addition, the DN T cells that were described previously express predominantly V14⁺Vß8.2⁺ TCRs rather than the V4.4⁺Vß5⁺ TCRs we observe among DN T cells in old B6 mice, further suggesting that Vß5⁺ DN T cells in B6 mice are distinct from previously described ßTCR⁺ DN T cells 51 .

Our ability to study Vß5⁺ T cells in this mouse model system has allowed us to identify three distinct pathways of tolerance induction in B6 mice and their role in shaping the CD4⁺, CD8⁺, and DN T cell compartments of mature animals. It is likely that similar pathways occur among other T cell subsets in response to a variety of Ags; however, the resulting changes in the peripheral T cell pool may be difficult to distinguish within a diverse T cell population. To extend these studies further, we examined the TCR V repertoire of the various subsets. Both the documentation of TCR-chain involvement in SAg recognition in other systems 52, 53, 54, 55, 56, 57, 58 and the inefficient formation of CD8^low cells among V2⁺Vß5⁺ T cells from B6.OT-1 Tg mice (Fig. 4) hint that the TCR V-chains expressed by Vß5 Tg mice influence tolerance induction. Age-related changes in the V repertoire could explain the increase in both the CD4:CD8 ratio and the percentage of peripheral CD4⁺ T cells expressing Vß5 in Tg mice aged 50–60 wk (Fig. 2). For instance, CD4⁺ T cells bearing V-chains that contribute to poor SAg interaction when paired with Vß5 52, 53, 54, 55, 56, 57, 58 could selectively survive or expand during old age, while the more SAg-reactive CD4⁺ T cells would be deleted, accounting for the upswing in the CD4:CD8 ratio and the percentage of CD4⁺ T cells expressing Vß5.

In many cases, the TCR V repertoire follows the anticipated pattern. Young mice (8–10 wk) are beginning to delete their CD4⁺ T cells (Figs. 1 and 2), and the increased expression of V3, V9/10, and V13 in Tg relative to non-Tg mice (Table III) may reflect SAg-mediated expansion of cells bearing these V families. These same V families are under-represented among CD4⁺ T cells from middle-aged and old Tg mice relative to non-Tg mice, perhaps because most of the CD4⁺ T cells bearing these SAg-responsive V elements are efficiently deleted with age. The over-representation of V4.4 and V5 among CD4⁺ T cells in old Tg animals may reflect poor interaction with vSAG8 and vSAG9, which allows T cells bearing these -chains to survive in old animals (Table III).

The V repertoire of CD8⁺ T cells in Vß5 Tg and non-Tg B6 mice shows many of the same trends as does the V repertoire of CD4⁺ T cells. One similarity is the poor expression of all V family members in middle aged Tg but not non-Tg animals (Table III), which could result from increased apoptosis and RNA degradation among the largely anergic population of Vß5⁺ T cells in middle-aged mice. Expression of the V7 family and the V4.4 gene are increased in CD8⁺ T cells from old Tg relative to old non-Tg animals, implying that T cells bearing these V-chains are resistant to becoming CD8^low cells and being deleted. To gain further insight into the induction of tolerance among CD8⁺ T cells, we also compared the V repertoire between CD8^low and CD8^high cells sorted from Vß5 Tg mice aged 15–18 wk. Sorting for CD8^low cells was necessary because these deletional intermediates do not survive the in vitro culture period 17 . Furthermore, their characterization allows us to study cells that are undergoing tolerance induction rather than those cells that are left behind. As expected from the data in Fig. 4, V2 expression was almost fivefold lower in CD8^low than in CD8^high cells, implying that V2, as well as V13 and V18, interact poorly with the tolerogen (Table IV). On the other hand, the increased expression of V9/10, V4.4, and V5 among CD8^low relative to CD8^high cells implies that T cells bearing these V-chains interact efficiently with the non-vSAG tolerogen and are more likely to be driven into the CD8^low compartment.

Finally, we analyzed the V repertoire of DN T cells and discovered a dramatic over-representation of V4.4 among DN T cells in old Tg mice (Table V). A similar over-representation of V4.4⁺ T cells was seen among CD4⁺ and CD8⁺ T cells in old Tg mice (Table III), which, along with the suggestion that DN T cells arise from CD4⁺ T cells, implies that V4.4⁺Vß5⁺ CD4⁺ T cells may survive in old mice because their TCR interacts poorly with vSAG8 and vSAG9 52, 53, 54, 55, 56, 57, 58 . Therefore, multiple interactions of V4.4⁺Vß5⁺ CD4⁺ T cells with vSAG8 or vSAG9 may lead to coreceptor down-regulation and cell survival as DN T cells.

Overall, our analyses indicate that the V repertoires of both non-Tg and Vß5 Tg mice undergo significant age-related variations as a result of the chronic induction of peripheral tolerance (Fig. 5 and Tables III–V), suggesting that the T cell repertoire is continually modulated in vivo. While our data provide the first characterization of the TCR V repertoire in B6 mice, our finding that V6, V7, and V12 expression levels are low (data not shown) has been independently confirmed in B10, B10.BR, B10.Q, and C57L mice 59, 60 . Previously, modifications of the TCR repertoire have been difficult to detect in the context of heterogeneous peripheral T lymphocyte populations. With the exception of expansion of oligoclonal populations of CD8⁺ T cells in old mice 40 and a restriction in the diversity of the Vß-chain in TCR V Tg mice 61 , alterations in TCR Vß expression as a result of clonal expansion or deletion have not been noted. Previous studies of the V repertoire in mice 59, 60 and humans 62 quantitated the percentage of cells expressing a particular -chain at one age, while our comparison of V expression between T cell subsets highlights age-related changes in expression during tolerance induction.

In Vß5 Tg mice, the induction of tolerance affects three different cell populations in distinct ways. Most CD4⁺Vß5⁺ T cells are deleted by vSAG8 and vSAG9, but a V4.4⁺ subset may survive and give rise to a population of DN T cells. Others survive by down-regulation of Vß5, reexpression of recombination machinery, and rearrangement and expression of endogenous Vß genes 63 . CD8⁺Vß5⁺ T cells are driven through a CD8^low deletional intermediate by their encounter with a tolerogen not encoded by an endogenous Mtv. Each tolerance pathway results in significant modifications in the TCR repertoire expressed by that compartment. Although the thymus plays an important role in shaping the developing immune repertoire, age-related changes in expression of V families in both Vß5 Tg and non-Tg B6 mice indicate that the balance between self-tolerance and a diverse TCR repertoire is subject to consistent and dramatic adjustments in the lymphoid periphery.

	Acknowledgments

 
We thank D. Morris for the gift of the WLC-0 mice, A. Pullen and J. Goverman for the gift of V PCR primers and/or sequences, L. Hood for use of the phosphorimager, and A. Aderem for use of phosphorimaging analysis software. We also thank D. Wilson for maintaining the mouse colony, K. Allen and D. Coder for assistance with flow cytometry, and M. Kelley and M. Maurer for assistance in the early stages of this project. Finally, we thank our colleagues M. Bevan, C. McMahan, T. Boursalian, S. Dillon, and N. Henig for their critical reading of the manuscript.

	Footnotes

 
¹ This work was supported by Research Grant AG-13078 from the National Institutes of Health to P.J.F. and National Institutes of Health Training Grant DK07467 to B.J.G. C.A.B. has been supported by National Institutes of Health Medical Scientist Training Program Grant GM-07226, the Life and Health Insurance Medical Research Fund, and the Poncin Scholarship Fund.

² Address correspondence and reprint requests to Dr. Pamela J. Fink, Department of Immunology, School of Medicine, University of Washington, Box 357650, Seattle, WA 98195. E-mail address:

³ Abbreviations used in this paper: B6, C57BL/6; B6.Thy1.1, B6.PL-Thy-1^a/Cy; Class II^-/-, MHC class II^-/-; DN, double negative CD4^-CD8^-; HPRT, hypoxanthine phosphoribosyltransferase; LN, lymph node; Mtv, mouse mammary tumor virus; PE, phycoerythrin; SAg, superantigen; TC, TriColor; Tg, transgenic; WT, wild-type.

Received for publication October 13, 1998. Accepted for publication December 4, 1998.

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