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The Journal of Immunology, 2003, 171: 6290-6296.
Copyright © 2003 by The American Association of Immunologists

Differential Regulation of Peripheral CD4+ T Cell Tolerance Induced by Deletion and TCR Revision 1

Mohamed Ali, Michael Weinreich, Stephanie Balcaitis, Cristine J. Cooper and Pamela J. Fink2

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In V{beta}5 transgenic mice, mature V{beta}5+CD4+ T cells are tolerized upon recognition of a self Ag, encoded by a defective endogenous retrovirus, whose expression is confined to the lymphoid periphery. Cells are driven by the tolerogen to enter one of two tolerance pathways, deletion or TCR revision. CD4+ T cells entering the former pathway are rendered anergic and then eliminated. In contrast, TCR revision drives gene rearrangement at the endogenous TCR {beta} locus and results in the appearance of V{beta}5-, endogenous V{beta}+, CD4+ T cells that are both self-tolerant and functional. An analysis of the molecules that influence each of these pathways was conducted to understand better the nature of the interactions that control tolerance induction in the lymphoid periphery. These studies reveal that deletion is efficient in reconstituted radiation chimeras and is B cell, CD28, inducible costimulatory molecule, Fas, CD4, and CD8 independent. In contrast, TCR revision is radiosensitive, B cell, CD28, and inducible costimulatory molecule dependent, Fas and CD4 influenced, and CD8 independent. Our data demonstrate the differential regulation of these two divergent tolerance pathways, despite the fact that they are both driven by the same tolerogen and restricted to mature CD4+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The immune system is charged with the dual tasks of protection against invading pathogens and preservation of self. In the predominant TCR {alpha}{beta} T cell compartment, it is within the thymus that Ag receptor diversity, MHC restriction specificity, and self-tolerance are first established (reviewed in Ref.1). However, it is now clear that emigration from the thymus does not signal the cessation of processes that mold the T cell population into one that is best suited to carry out the tasks it has been assigned. Homeostasis and survival of naive T cells depend on basal signaling by the TCR, triggered by its interaction with MHC and peptide (2, 3). Thus, MHC restriction specificity continues to be tested long after the T cell has graduated from the thymic microenvironment. Similarly, the TCR repertoire, first established in developing T cells by somatic recombination, can be further modified in the periphery (4, 5, 6, 7, 8, 9). Tolerance induced to self Ags first encountered by mature T cells outside the thymus occurs by multiple mechanisms, including nutrient deprivation (10), active suppression (11), and anergy and deletion (12).

We have analyzed the induction of tolerance among mature T cells to an endogenous superantigen encoded by a defective retrovirus, mouse mammary tumor virus 8 (Mtv-8). 3 This tolerogen interacts weakly with cells expressing V{beta}5+ TCRs, and is unusual among superantigens in failing to drive significant levels of T cell proliferation in vitro (13, 14). Mtv-8 is carried by most strains of laboratory mice, including C57BL/6 (B6), and as a result of methylation, its expression is weak (15) and limited to the lymphoid periphery (16, 17). Analysis of V{beta}5 transgenic (Tg) B6 mice has defined two distinct pathways that characterize the induction of tolerance in mature CD4+ T cells to this self Ag (18). The majority of V{beta}5+CD4+ T cells become anergic and are deleted, resulting in a prominent age-dependent inversion of the peripheral CD4:CD8 ratio in these animals (19, 20, 21). Some of the remaining V{beta}5+CD4+ T cells from both V{beta}5 Tg and non-Tg B6 mice undergo a novel process that results in cell rescue and the appearance of a population of diverse, self-tolerant T cells. This alternate pathway, termed TCR revision, is characterized by sequential loss of V{beta}5 surface expression, induction of the lymphocyte-specific components of the recombinase machinery (including recombination-activating genes (RAG)1, RAG2, and TdT), and rearrangement of endogenous TCR {beta}-chain genes (4, 5). TCR revision does not solely target recent thymic emigrants, but can induce TCR rearrangement in cells that have emigrated from the thymus at least 2 mo previously (9). The newly generated TCR repertoire is self-tolerant, functional, and nearly as diverse as the non-Tg T cell repertoire at both the molecular and cellular levels (5).

It is striking that deletion and TCR revision are both restricted to CD4+ T cells in V{beta}5 Tg mice, given the fact that cells traveling down each pathway experience such diametrically opposed outcomes: death vs survival. In an effort to understand better how these two distinct pathways are regulated, the present study was undertaken to define the molecules that influence them. Our analyses demonstrate that while deletion is efficient in reconstituted radiation chimeras and is B cell, CD28, inducible costimulatory molecule (ICOS), Fas, CD4, and CD8 independent, TCR revision recovers slowly in radiation chimeras and is B cell, CD28, and ICOS dependent, Fas and CD4 influenced, and CD8 independent. Our results suggest that these two divergent tolerance pathways are differentially regulated, despite the fact that they are both driven by the same tolerogen and restricted to the same population of mature T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

B6 and B6.PL-Thy-1a/Cy (B6.Thy-1.1) mice were purchased from The Jackson Laboratory (Bar Harbor, ME), and (B6 x B6.Thy-1.1)F1 mice were bred in a specific pathogen-free barrier facility at the University of Washington. V{beta}5 TCR Tg mice were generated on a B6 background (19) and maintained in our facility as heterozygotes by crossing TCR Tg with B6 mice and screening PBLs for surface expression of V{beta}5. Fas-deficient V{beta}5 Tg mice were generated by cross/backcross breeding of V{beta}5 Tg and B6.MRL-Faslpr (B6.lpr) mice (purchased from The Jackson Laboratory), and screened for V{beta}5 expression by flow cytometry and for the lpr mutation by PCR (22). The V{beta}5 transgene was crossed onto the µMT null (IgH null) B6 mice from The Jackson Laboratory (23), and weanlings were screened for transgene expression and the loss of B220+ B cells.

B6-Cd28tm1Mak (CD28 null B6 mice) were purchased from The Jackson Laboratory (24), and the V{beta}5 transgene was crossed onto this background. PBLs from weanlings were screened for surface expression of V{beta}5 and for the neomycin insertion into the CD28 locus by PCR of tail DNA using the forward primer, 5'-GCCCGGTTCTTTTTGTCAAGACCGA and the reverse primer, 5'-ATCCTCGCCGTCGGGCATGCGCGCC for 35 cycles at 72°C. Wild-type CD28 alleles were detected with the forward primer, 5'-TTGGTAAAGCAGTCGCCCCTGCTT and the reverse primer, 5'-AGTTCCATTGCTCCTCTCGTTGTC. Reaction products were electrophoresed through 2% agarose. With the relevant primers, the wild-type CD28 allele gives a product of 310 bp, and the CD28 null allele gives a product of 436 bp.

The V{beta}5 transgene was crossed onto B6 mice Tg for CD8.1 under the control of the human CD2 promoter that was obtained from E. Robey (University of California, Berkeley, CA) (25). PBLs were screened for surface expression of V{beta}5 and CD8.1. CD4 null B6 mice carrying a human CD2 transgene under the control of the CD4 promoter (26) were obtained from B. Fowlkes (National Institutes of Health, Bethesda, MD). The V{beta}5 transgene was crossed onto this background, and PBLs from weanlings were screened by flow cytometry for the absence of CD4+ cells and for surface expression of human CD2 and V{beta}5. The V{beta}5 transgene was crossed onto ICOS null B6 mice that were obtained from C. Dong (University of Washington, Seattle, WA) (27). PBLs were screened by flow cytometry for expression of V{beta}5 and by PCR for both the wild-type and targeted mutation in the ICOS gene using the ICOS forward primer, 5'-ACCCTCATCCATGCAGTGATTC with either the wild-type ICOS reverse primer, 5'-GGCTACAGAATGAGTTGCACAAG or the neomycin reverse primer, 5'-CTCCAGACTGCCTTGGGAAAA. Using a touchdown program on DNA engine from MJ Research (Waltham, MA), the former reaction amplifies a 750-bp wild-type band, and the latter a 760-bp product from the null allele.

Mice used in these experiments were all either generated on a B6 background or backcrossed a minimum of 10 generations onto this background, and all carry Mtv-8. This defective retrovirus encodes the self Ag that drives both deletion and TCR revision among mature peripheral CD4+ T cells in V{beta}5 Tg mice (21). All experiments were performed in compliance with the guidelines of the University of Washington Institutional Animal Care and Use Committee.

Construction of radiation bone marrow chimeras

Radiation chimeras were generated, as previously described (28). Briefly, (B6 x B6.Thy-1.1)F1 recipients were administered 925 rad from a 137Cs source and injected i.v. 6 h later with 107 T cell-depleted bone marrow cells from sex-matched V{beta}5 Tg B6 (Thy-1.2+) donors. These animals are designated V{beta}5 Tg->B6 radiation chimeras. At the indicated time points, after irradiation and reconstitution, PBLs were stained for V{beta}5, CD4, and CD8, and for Thy-1.1 and Thy-1.2 to distinguish donor- from host-derived cells.

Abs, cell surface staining, and flow cytometric analysis

Splenocytes and PBL obtained from water-lysed blood were resuspended in HBSS plus 1% BSA for staining. Flow cytometric analyses were performed using CellQuest software on either a FACSCalibur or a FACScan flow cytometer (BD Biosciences, Mountain View, CA). A minimum of 100,000 live-gated events was analyzed per sample. The following Abs were purchased from BD PharMingen (San Diego, CA): FITC-conjugated anti-human CD2 (RPA-2.10), anti-V{beta}5 (MR9-4), anti-TCR {beta} (H57-597), anti-Thy-1.2 (30H12), PE-conjugated anti-CD45R/B220 (RA3-6B2), anti-CD4 (RM4-5), anti-V{beta}5 (MR9-4), biotin-conjugated anti-Thy-1.1 (HIS51), anti-Thy-1.2 (30H12), anti-CD8.2 (53-5.8), PerCP-conjugated anti-CD8{alpha} (53-6.7), allophycocyanin-conjugated anti-CD4 (RM4-5), and anti-Thy-1.2 (30-H12). FITC-conjugated anti-CD8.1 (CD8-E1) was purchased from American Research Products (Belmont, MA). Quantum red-conjugated anti-CD4 (H129.19) was purchased from Sigma-Aldrich (St. Louis, MO). Tricolor-conjugated anti-CD8 (CT-CD8{alpha}) and streptavidin were purchased from Caltag Laboratories (Burlingame, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Radiation and bone marrow reconstitution differentially influence the deletion of V{beta}5+CD4+ and the accumulation of V{beta}5-CD4+ peripheral T cells in V{beta}5 Tg mice

F1(B6 x B6.Thy-1.1) recipients were irradiated and reconstituted with bone marrow from congenic V{beta}5 Tg B6 donors, and the extent of deletion and loss of transgene expression within the peripheral CD4+ T cell compartment were measured. The deletion of V{beta}5+CD4+ T cells, evidenced as a decline in the CD4:CD8 ratio (19, 20, 21), proceeds to the same extent in radiation chimeras as in unmanipulated transgenics, but with somewhat slower kinetics (Fig. 1A). In contrast, the accumulation of V{beta}5-CD4+ peripheral T cells, a measure of TCR revision (4, 5, 9), is barely detectable in radiation chimeras until nearly a full year after their generation (Fig. 1, B and C). Although irradiation and reconstitution impact many immunological functions, these data suggest that deletion and TCR revision among mature peripheral CD4+ T cells are differentially regulated, despite the fact that both tolerance pathways are Mtv-8 dependent and restricted to mature peripheral CD4+ T cells (21). These experiments gave impetus to the search for molecules whose expression impacts deletion and revision in qualitatively or quantitatively distinct ways.



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  		FIGURE 1. Radiation sensitivity helps distinguish deletion and TCR revision as separate pathways of Mtv-8-driven tolerance induction among mature V{beta}5+CD4+ T cells. PBLs from B6 and V{beta}5 Tg mice at the indicated ages and from V{beta}5 Tg->(B6 x B6.Thy-1.1) radiation chimeras (labeled as V{beta}5 Tg->B6) at the indicated times after irradiation and reconstitution were stained with anti-Thy-1.1, anti-V{beta}5, anti-Thy-1.2, and either anti-CD8 or anti-CD4 Abs. A, The CD4:CD8 ratio among peripheral T cells inverts to the same extent, but with somewhat slower kinetics in radiation chimeras compared with unmanipulated V{beta}5 Tg mice. The mean CD4:CD8 ratio among peripheral Thy-1.1-Thy-1.2+ cells is plotted vs age for three to seven non-Tg B6 mice (filled bars) and four to seven V{beta}5 Tg B6 mice (shaded bars), and vs weeks postreconstitution for three to five radiation chimeras (stippled bars) within each age group. Error bars designate the SEMs. B, Representative dot plots of splenocytes gated on CD4+Thy-1.2+Thy-1.1- cells from V{beta}5 Tg mice 20 and 40 wk of age and from V{beta}5 Tg->B6 radiation chimeras 20 and 40 wk after reconstitution. Data for the two time points were collected on different days. C, The loss of V{beta}5 expression among peripheral CD4+ T cells occurs with slower kinetics and to a lesser extent in radiation chimeras compared with unmanipulated V{beta}5 Tg mice. Live gated Thy-1.1-Thy-1.2+CD4+ donor cells were analyzed for V{beta}5 expression. Transgene expression among CD8+ T cells in each sample remained above 97% at all time points. The mean percentage of CD4+ peripheral T cells that are V{beta}5+ is plotted against age or weeks postreconstitution for the V{beta}5 Tg mice (shaded bars) and radiation chimeras (stippled bars) described in A. Error bars designate the SEMs.

 
Fas is not required for the deletion of V{beta}5+CD4+ T cells, but loss of Fas expression dramatically enhances the accumulation of V{beta}5-CD4+ T cells

V{beta}5 Tg B6.lpr mice carry an insertional mutation in the gene encoding the death-inducer Fas (CD95). These mice were analyzed to determine whether Fas-mediated death is involved in either deletion or TCR revision during the induction of tolerance to Mtv-8. The peripheral CD4:CD8 ratio inverts in V{beta}5 Tg mice on an lpr background with kinetics similar to those in wild-type V{beta}5 Tg B6 mice. The ratio is lower in 10- to 30-wk-old Tg mice expressing mutant Fas than in age-matched wild-type mice, most likely a reflection of the slightly lower CD4:CD8 ratio in non-Tg B6.lpr relative to B6 mice (Fig. 2A). In contrast, loss of Fas expression is associated with an enhanced accumulation of CD4+ T cells that have lost transgene expression. The appearance of V{beta}5-CD4+ T cells, shown previously to be the end products of TCR revision (4, 5, 9), is both accelerated and exaggerated in V{beta}5 Tg B6.lpr mice (Figs. 2, B and C, and 5B). Thus, Fas-mediated death is not essential for the deletion of Mtv-8-reactive V{beta}5+CD4+ T cells, but may be involved in trimming the population of transgene-negative CD4+ peripheral T cells.



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  		FIGURE 2. Fas-mediated death is not required for the Mtv-8-dependent deletion of peripheral V{beta}5+CD4+ T cells, but does influence the accumulation of V{beta}5-CD4+ peripheral T cells in V{beta}5 Tg mice. Splenocytes from mice 3 wk of age and younger and PBL from mice older than 3 wk were stained with anti-V{beta}5, anti-CD4, and anti-CD8 Abs. A, V{beta}5+CD4+ T cells are deleted in V{beta}5 Tg B6.lpr mice. The peripheral CD4:CD8 ratio is plotted against weeks of age for individual non-Tg B6 ({circ}), non-Tg B6.lpr ({blacktriangleup}), V{beta}5 Tg B6 (•), and V{beta}5 Tg B6.lpr mice ({triangleup}). Data from lpr heterozygotes and homozygotes were indistinguishable and were combined. B, Representative dot plots of splenocytes gated on CD4+ cells from V{beta}5 Tg B6.lpr mice 11 and 37 wk of age. The percentage of V{beta}5+ of total CD4+ cells is indicated. C, V{beta}5-CD4+ T cells accumulate more rapidly and to a greater extent in V{beta}5 Tg B6.lpr mice than in their wild-type V{beta}5 Tg counterparts. The percentage of peripheral cells that are V{beta}5+ is plotted vs age in weeks for CD8+ T cells from V{beta}5 Tg B6 ({circ}), CD4+ T cells from V{beta}5 Tg B6 ({blacksquare}), and CD4+ T cells from V{beta}5 Tg B6.lpr mice ({triangleup}). Transgene expression in CD8+ T cells from V{beta}5 Tg B6.lpr mice remained above 95% in mice of all age groups.

 
CD8 expression by CD4+ T cells does not prevent deletion of CD4+V{beta}5+ cells or TCR revision

Mtv-8-dependent deletion of peripheral T cells and the loss of transgene expression that results from TCR revision in V{beta}5 Tg mice are both restricted to the CD4+ population. It was therefore of interest to test whether aberrant expression of CD8 by CD4 lineage cells alters their sensitivity either to deletion or TCR revision. The V{beta}5 transgene was crossed onto a CD8.1 Tg B6 (CD8.2+) background, such that both CD4+ and CD8.2+ T cells from these mice express the CD8.1 transgene. The ratio of CD4:CD8.2 peripheral T cells from the CD8.1 Tg V{beta}5 Tg mice decreases with the same kinetics and to the same extent as that in CD8.1 non-Tg V{beta}5 Tg mice (Fig. 3A). It is interesting that the CD4:CD8.2 ratio in CD8.1 Tg V{beta}5 non-Tg mice in all age groups is significantly lower than that in CD8.1 non-Tg V{beta}5 non-Tg mice (see Discussion). Further analyses of these double Tg mice revealed that the accumulation of V{beta}5-CD4+ T cells is indistinguishable in CD8.1 Tg and non-Tg mice (Fig. 3B). Thus, expression of CD8 by CD4+ T cells does not interfere with either Mtv-8-dependent deletion of V{beta}5+CD4+ mature T cells or the efficiency with which they undergo TCR revision.



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  		FIGURE 3. CD8 expression by CD4+ V{beta}5 Tg T cells does not prevent their deletion nor alter their age-dependent loss of V{beta}5 transgene expression. Splenocytes from mice 3 wk of age and younger and PBL from mice older than 3 wk were stained with anti-V{beta}5, anti-CD8.1, anti-CD4, and anti-CD8.2. In the samples from CD8.1 Tg donors, CD4 lineage cells were defined as CD8.1+CD8.2-CD4+; CD8 lineage cells were defined as CD8.1+CD8.2+CD4-. A, V{beta}5+CD4+ T cells are deleted in V{beta}5 Tg CD8.1 Tg mice. The mean peripheral CD4:CD8.2 ratios of cells from mice of the indicated age ranges are plotted vs age for CD8.1 non-Tg V{beta}5 non-Tg (filled bars, n = 9–23 for all age groups), CD8.1 Tg V{beta}5 non-Tg (open bars, n = 9–18 for all age groups), CD8.1 non-Tg V{beta}5 Tg (shaded bars, n = 10–33 for all age groups), and CD8.1 Tg V{beta}5 Tg (striped bars, n = 15–38 for all age groups). Error bars designate the SEMs. Using a two-tailed, Student’s t test with equal variance, the ratios of CD8.1 non-Tg and CD8.1 Tg V{beta}5 non-Tg mice are significantly different, with p values of 8.5 x 10-4, 3.5 x 10-3, .02, and 9.9 x 10-3 for mice 6–15, 16–25, 26–35, and 36–45 wk of age, respectively. A similar comparison shows the ratios are not significantly different between CD8.1 non-Tg and CD8.1 Tg V{beta}5 Tg mice within these same age ranges. B, V{beta}5-CD4+ T cells accumulate to the same extent in V{beta}5 Tg CD8.1 Tg mice as in their CD8.1 non-Tg counterparts. The percentage of CD4+ peripheral T cells that are V{beta}5+ is plotted vs age in weeks for individual CD8.1 Tg V{beta}5 Tg ({circ}) and CD8.1 non-Tg V{beta}5 Tg mice ({blacksquare}).

 
CD4 expression by CD4 lineage T cells is not required for deletion of V{beta}5+CD4+ peripheral T cells, but may facilitate TCR revision

To determine whether CD4 expression is essential for the dual tolerance processes that target CD4+ peripheral T cells in Mtv-8+ V{beta}5 Tg mice, the V{beta}5 transgene was crossed onto a CD4 null background, in which CD4 lineage cells can be tracked by their expression of human CD2, driven by the CD4 promoter (26). As expected, very few CD4 lineage cells (defined in this work as CD8-CD2+Thy-1.2+ cells) develop in these animals. Despite these low numbers, it is clear that deletion of CD4 lineage peripheral T cells occurs in V{beta}5 Tg relative to non-Tg mice (Fig. 4A). The ratios in V{beta}5 Tg and non-Tg CD4 null animals 2 wk of age and younger are indistinguishable, as is the case in CD4 wild-type mice (Fig. 2A). After this age, the ratio in V{beta}5 Tg CD4 null mice falls below that seen in their non-Tg littermates, and remains lower. In contrast, while V{beta}5-CD4 lineage cells accumulate in CD4 null animals, they do so to a lesser extent than in CD4 wild-type mice (Fig. 4B). Thus, CD4 expression is not required for the selective deletion of CD4 lineage cells in Mtv-8+ V{beta}5 Tg mice, but the loss of CD4 expression alters the tempo and extent of TCR revision, as measured by the accumulation of its V{beta}5-CD4 lineage end products.



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  		FIGURE 4. Loss of CD4 expression by CD4 lineage T cells in V{beta}5 Tg mice does not interfere with deletion of V{beta}5+ cells, but diminishes the tempo and extent of accumulation of V{beta}5-CD4 lineage T cells. Splenocytes from mice 3 wk of age and younger and PBLs from mice older than 3 wk were stained with anti-human CD2, anti-V{beta}5, anti-CD8, and anti-Thy-1.2. In the samples from the CD4 null donors, CD4 lineage cells were defined as CD8-CD2+Thy-1.2+ cells. A, CD4 null CD4 lineage cells are deleted in V{beta}5 Tg mice. The ratio of CD4 lineage to CD8+ peripheral T cells is plotted vs age for individual V{beta}5 Tg CD4 null B6 ({blacksquare}) and V{beta}5 non-Tg CD4 null B6 mice ({triangleup}). B, Transgene expression is lost, but to a lesser extent, in CD4 null compared with CD4 wild-type V{beta}5 Tg B6 mice. The percentage of CD4 lineage peripheral T cells that are V{beta}5+ is plotted vs age for individual V{beta}5 Tg CD4 null B6 ({blacksquare}) and V{beta}5 Tg CD4 wild-type (wt) B6 mice ({triangleup}).

 
Loss of ICOS expression does not interfere with the deletion of V{beta}5+CD4+ T cells, but greatly retards the accumulation of V{beta}5-CD4+ T cells

ICOS expression is known to influence the formation of germinal centers (GCs) (29), one compartment in which TCR revision may occur (4, 18, 30). It was therefore of interest to determine the differential influence of ICOS expression on deletion and TCR revision in V{beta}5 Tg mice. The deletion of CD4+ T cells in V{beta}5 Tg ICOS null mice occurs to the same extent and with the same kinetics as in ICOS wild-type mice (Fig. 5A). In contrast, the accumulation of V{beta}5-CD4+ T cells in V{beta}5 Tg ICOS null mice is delayed by ~10 mo relative to that in ICOS wild-type mice (Fig. 5B, and data not shown). This impressive delay is similar to that seen in both V{beta}5 Tg CD28 null and B cell null animals (4, 5) (Fig. 5B). Thus, ICOS dependence is a trait that distinguishes Mtv-8-dependent TCR revision and deletion within the V{beta}5+CD4+ mature T cell population.



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  		FIGURE 5. V{beta}5+CD4+ T cell deletion occurs normally in ICOS null V{beta}5 Tg mice, but accumulation of V{beta}5-CD4+ T cells is greatly retarded. Splenocytes from mice 3 wk of age or younger and PBLs from mice older than 3 wk were stained with anti-V{beta}5, anti-CD4, and anti-CD8 Abs. A, The peripheral CD4:CD8 ratio is plotted as a function of age for individual non-Tg B6 ({circ}), V{beta}5 Tg B6 (•), non-Tg ICOS null ({blacktriangleup}), and V{beta}5 Tg ICOS null mice ({triangleup}). B, The percentage of CD4+ peripheral T cells that are V{beta}5+ is plotted as a function of the indicated age range for V{beta}5 Tg mice of the following backgrounds: B6 (filled bars, n = 24–39), lpr (stippled bars, n = 29–35), B cell null (open bars, n = 17–26), CD28 null (shaded bars, n = 6–18), and ICOS null (striped bars, n = 16–22). Error bars designate the SEMs. Using the two-tailed Student’s t test with equal variance, the percentage of V{beta}5+ of CD4+ T cells in V{beta}5 Tg B6 mice differs significantly from that in V{beta}5 Tg B6.lpr mice, with p values of 4 x 10-6, 2.9 x 10-7, 1.1 x 10-3, and 0.01 for mice 6–16, 16–25, 26–35, and 36–50 wk of age, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In V{beta}5 TCR Tg mice, tolerance induced to the self Ag encoded by the defective endogenous retrovirus Mtv-8 occurs through two distinct mechanisms, deletion and TCR revision. Although both pathways target mature CD4+ T cells, deletion results in the age-dependent inversion of the CD4:CD8 ratio in the lymphoid periphery, and TCR revision drives the generation of a diverse population of self-tolerant, functional, endogenous V{beta}-expressing, surface V{beta}5- T cells (4, 5, 9, 19, 20, 21). The existence of two distinct tolerance pathways was first suggested by the observation that the accumulation of V{beta}5-CD4+ T cells in V{beta}5 Tg mice is severely impaired in B cell null mice, while deletion of V{beta}5+CD4+ T cells in the same animals occurs unabated (4). Using this observation as a foundation, the present studies were conducted to expand our understanding of the differential regulation of these two tolerance mechanisms. As summarized in Fig. 6, the data presented in this work reveal that Mtv-8-driven deletion of V{beta}5+CD4+ peripheral T cells is independent not only of B cells, but also of Fas, CD4, CD8, ICOS, and CD28 expression, and recovers quickly and completely after lethal irradiation and bone marrow reconstitution. In sharp contrast, the efficiency of TCR revision is strongly influenced by Fas and CD4 expression, is dependent on the presence of B cells and on ICOS and CD28 expression, and recovers very slowly and only partially after irradiation and reconstitution (Fig. 6).



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  		FIGURE 6. Model depicting CD4+ T cell tolerance induction to Mtv-8 in V{beta}5 Tg mice. TCR revision is characterized by the loss of V{beta}5 surface expression, induction of the lymphocyte-specific components of the recombinase machinery, rearrangement at endogenous TCR {beta} loci, and subsequent expression of endogenous TCRs by functional, self-tolerant CD4+ T cells. This process recovers poorly in radiation chimeras and is B cell, ICOS, and CD28 dependent. CD4 lineage cells that lack CD4 expression are inefficient at completing TCR revision, while failure of a CD4+ T cell to express a functional Fas molecule improves its chances of surviving this process. Expression of CD8 by CD4 lineage T cells does not influence TCR revision. In contrast, Mtv-8-dependent deletion of CD4+ T cells occurs efficiently in radiation chimeras and is B cell, ICOS, CD28, CD4, Fas, and CD8 independent. These clear distinctions between deletion and TCR revision, both tolerance mechanisms that are Mtv-8 mediated and restricted to CD4+ mature peripheral T cells, further define these pathways and illustrate their differential regulation.

 
Precise identification of Mtv-expressing cells has been impeded by the low level of endogenous superantigen expression and by the difficulty in generating Mtv-specific Abs (reviewed in Ref.13). Only a few observations provide helpful information in this regard. Mtv-8 expression appears to be exceptionally weak (15, 31), largely restricted to lymphoid cells (16, 32), and limited to the periphery (17). Furthermore, Mtv-8-dependent deletion of V{beta}5+ T cells also occurs in the periphery of non-Tg B6 mice (19, 33). Surface expression of endogenous superantigens is restricted to MHC class II-positive cells, and is positively regulated by MHC class II expression levels (13, 34). Endogenous superantigen-expressing cells include, but are not limited to B cells (16, 32), thought to be the most potent stimulators of Mtv-driven T cell proliferation (reviewed in Ref.13). The fact that deletion occurs uninterrupted in B cell null V{beta}5 Tg mice (4) indicates that cells other than B cells are capable of expressing Mtv-8 in a tolerogenic form, consistent with data from other superantigen systems (35, 36). The heightened radiation sensitivity of TCR revision relative to deletion is also interesting in this regard. Deletion generally follows the reconstitution kinetics of the hemopoietic compartment; the inversion of the CD4:CD8 ratio in chimeric mice is delayed by ~2 mo from that of unirradiated V{beta}5 Tg mice (Fig. 1A). In contrast, the accumulation of V{beta}5-CD4+ T cells is delayed by ~9 mo, suggesting that the relevant cellular compartments are radiation sensitive, but only slowly reconstituted upon bone marrow engraftment. One such compartment is that of follicular dendritic cells, the class II-positive APCs in GCs, whose formation is radiosensitive (37, 38). This correlation is interesting in light of other evidence consistent with a GC localization for TCR revision (see below). It is also likely that other aspects of the peripheral environment contribute to the differential radiosensitivity of TCR revision relative to deletion.

Our findings that Fas-mediated death is not essential for Mtv-8-driven deletion of V{beta}5+CD4+ T cells (Fig. 2A) are consistent with previous data demonstrating the Fas independence of tolerance to other endogenous superantigens (39, 40). Perhaps more interesting is the finding that loss of Fas expression exaggerates the accumulation of the V{beta}5-CD4+ end products of TCR revision (Fig. 2C). Again, there are parallels in the GC. B cells up-regulate Fas expression in the GC, and Fas-mediated death plays a role in the selection of B cells in the GC reaction, as both clonal selection and affinity maturation are disregulated in B6.lpr mice (reviewed in Ref.41). Furthermore, up to 20% of post-GC-derived B cell lymphomas carry mutations in the gene encoding Fas (reviewed in Ref.42). CD4+ T cells are also known to be sensitive to Fas-mediated death pathways (43, 44, 45). Drawing a parallel, Fas-mediated death may also play a role in selection of T cells undergoing TCR revision, as in its absence, more V{beta}5-CD4+ T cells are allowed to accumulate. It is also possible, although we feel less likely, that cross-linking Fas on the surface of V{beta}5-CD4+ T cells causes their death, while cross-linking Fas on the surface of V{beta}5+CD4+ T cells causes their proliferation (46). The loss of Fas in lpr mice could thereby drive the increase in the V{beta}5- relative to the V{beta}5+ population.

To test the influence of coreceptor expression on TCR revision, CD8+CD4 lineage cells were analyzed for their susceptibility to this tolerance mechanism. Our data show that aberrant expression of CD8 on CD4 lineage T cells does not alter their susceptibility to either Mtv-8-driven deletion or TCR revision (Fig. 3). This aberrant expression is also unlikely to impede the access of CD4+ T cells to GCs, as this migration appears to be chemokine mediated, and therefore unlikely to be influenced by coreceptor expression (47, 48). On a side note, the CD4:CD8.2 ratio in CD8.1 Tg V{beta}5 non-Tg B6 mice is significantly lower than that in double non-Tg B6 mice, in all age groups (Fig. 3A, compare filled with open bars). Given that CD4+ and CD8+ T cells represent partially overlapping niches (49), these data suggest that the expression of CD8 by CD4 lineage T cells may give them a CD8-like appearance to the homeostasis machinery, which then maintains this population at the lower levels characteristic of CD8 lineage cells.

On the flip side of the question of coreceptor expression and tolerance induction, we asked whether the absence of CD4 expression by CD4 lineage cells impacts their sensitivity to deletion or TCR revision mediated by Mtv-8. Although it is clear that CD4 lineage T cells are deleted with kinetics similar to those in CD4 wild-type V{beta}5 Tg mice (Fig. 4A), the small numbers of such cells in CD4 null mice prevent comparison of the extent of their deletion in CD4-sufficient and null mice. Unperturbed Mtv-8-mediated deletion in CD4 null mice is consistent with previous findings that deletion of T cells recognizing other endogenous superantigens can occur on a CD4 null background (50, 51). In contrast, it is very clear that a smaller fraction of CD4 lineage cells lose V{beta}5 expression in CD4 null compared with CD4 wild-type mice (Fig. 4B). There are several possible explanations for the relationship between CD4 expression and the accumulation of TCR revision end products. CD4 expression may influence the ability of a cell to enter the lymphoid compartment in which TCR revision takes place. In addition, the absence of a class II-binding coreceptor will modulate the signal delivered upon recognition of Mtv-8 on a class II-positive presenting cell, and this perturbation may result in inefficient TCR revision. However, it is perhaps equally likely that the defective thymic selection that results from such aberrant loss of coreceptor expression will influence the TCR {alpha}-chain repertoire expressed by the few CD4 lineage T cells that have successfully differentiated in CD4 null V{beta}5 Tg mice. This repertoire most likely influences sensitivity to TCR revision (21).

Our data show that expression of the costimulatory molecule ICOS is necessary for efficient TCR revision, but does not appear to influence Mtv-8-mediated deletion of V{beta}5+ T cells (Fig. 5). The inversion of the peripheral CD4:CD8 ratio is indistinguishable in ICOS null and wild-type V{beta}5 Tg mice (Fig. 5A), while the accumulation of V{beta}5-CD4+ T cells is greatly delayed in the former compared with the latter mice (Fig. 5B). The restriction of ICOS expression to activated and GC T cells (29, 52, 53) again suggests the involvement of GCs in Mtv-8-dependent TCR revision, but not deletion. This notion is strengthened by our previous findings that TCR revision is selectively diminished in mice lacking B cells (4) and CD28 molecules (5). In all three types of mice, GC formation is severely diminished (29, 54). An additional, nonmutually exclusive explanation for the near ablation of TCR revision in mice lacking B cells and ICOS and CD28 molecules is the possibility that B cells (known to express the ligands for ICOS and CD28) are selectively capable of delivering the signal that drives ICOS+CD28+ T cells to undergo TCR revision, regardless of where this revision takes place.

Altogether, our data provide strong evidence for the differential regulation of two distinct pathways for the induction of self-tolerance among Mtv-8-reactive, CD4+ peripheral T cells. Although deletion (and therefore superantigen expression) is independent of CD4, Fas, CD28, and ICOS expression, the efficiency of TCR revision is strongly influenced by these molecules. The strong correlation between efficient GC formation and TCR revision is striking. These observations, strengthened by the fact that the GC provides a microenvironment promoting selection for Ag receptor specificity within the B cell compartment, lead us to suggest that Mtv-8-driven TCR revision occurs within the GC.


   Acknowledgments
 
We thank Khristina Kline for her careful work in the early stages of this study, Phillip Zhang for invaluable help with animal husbandry, Dr. C. Dong for the ICOS null mice, Dr. B. J. Fowlkes for the CD4 null, human CD2 Tg mice, and Dr. E. Robey for the CD8.1 Tg mice.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AG13078 and AI 44130 (to P.J.F.) and the Juvenile Diabetes Research Foundation Chet Edmonson postdoctoral fellowship (to C.J.C.). Back

2 Address correspondence and reprint requests to Dr. Pamela Fink, Department of Immunology, University of Washington, Campus Box 357650, Seattle, WA 98195. E-mail address: pfink{at}u.washington.edu Back

3 Abbreviations used in this paper: Mtv, mammary tumor virus; GC, germinal center; ICOS, inducible costimulatory molecule; RAG, recombination-activating gene; Tg, transgenic. Back

Received for publication July 2, 2003. Accepted for publication September 30, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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