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Editing Autoreactive TCR Enables Efficient Positive Selection¹

Fabio R. Santori, Ivica Arsov², Mirjana Lili and Stanislav Vukmanovi³

Michael Heidelberger Division of Immunology, Department of Pathology and Kaplan Cancer Center, New York University School of Medicine, New York, NY 10016

	Abstract

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Allelic exclusion is inefficient at the TCR locus, allowing a sizeable portion of T cells to carry two functional TCRs. The potential danger of dual TCR expression is a rescue of autoreactive TCRs during selection in the thymus and subsequent development of autoimmunity. In this study, we examine the reason(s) for replacing an autoreactive TCR and for allowing the survival of cells carrying two TCRs. We compared development of TCR transgenic CD4⁺CD8^- thymocytes in the presence or absence of MHC class II autoantigen that does not induce deletion of thymocytes. Contrary to the expected negative effect of the presence of autoantigen, 100% more CD4⁺CD8^- thymocytes were found in the presence of MHC class II autoantigen than in the neutral background. A further increase in the strength of autoantigenic signal via expression of a human CD4 transgene led to an additional increase in the numbers of CD4⁺CD8^- thymocytes. Thus, editing autoreactive TCR results in more efficient positive selection, and this may be both a reason and a reward for risking autoimmunity.

	Introduction

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Rearrangement of the TCR loci is activated by the coordinate action of recombination-activating gene (RAG)⁴-1 and RAG-2 molecules and is characterized by random joining of one V, (D), and J gene elements (reviewed in Refs. 1 and 2). The TCR locus is rearranged at a developmentaly earlier stage than the TCR locus. Expression of functional TCR chain enables immature CD4^-CD8^-TCR^- thymocytes to pass through a developmental checkpoint and acquire CD4 and CD8 coreceptors (3, 4). TCR expression also results in temporary RAG down-modulation (5) and in allelic exclusion, that is, inaccessibility of the second germline TCR locus to recombination (6). At the subsequent stage of thymocyte differentiation (CD4⁺CD8⁺TCR^-), the TCR locus becomes accessible for recombination, and a second wave of RAG expression is induced (5). Productive TCR rearrangement results in expression of low levels of cell surface TCR. The resulting CD4⁺CD8⁺TCR^low population of thymocytes is then subject to two selection processes, both based on the interaction of their TCR with self MHC/peptide complexes. Positive selection is a result of relatively weak TCR engagement that delivers survival and differentiation signals to only a fraction of total CD4⁺CD8⁺ thymocytes (7). Failure to express the TCR or expression of TCR(s) not reactive with self peptide/MHC complexes leads to CD4⁺CD8⁺ thymocyte death by default (8). Thymocytes expressing TCRs with potentially harmful reactivity with self peptide/MHC (characterized by relatively strong interactions) must be silenced or eliminated. Negative selection can be achieved by physical deletion or by a variety of nondeletional mechanisms, including down-modulating the levels of coreceptor molecules, rendering cells anergic, or raising the activation thresholds in T cells (reviewed in Ref. 9).

Unlike in the TCR locus, allelic exclusion at the TCR locus is almost nonexistent (10). In fact, rearrangement at the TCR locus continues until signals for positive selection terminate RAG-1 and RAG-2 expression (11, 12). Residual levels of RAG-1 and RAG-2 from the first wave of expression are sufficient to initiate recombination at the TCR locus (13). However, in the absence of additional RAG expression, rearrangement is limited to the 5' portion of the J cluster of one locus (13). Thus, a secondary wave of RAG expression appears to be initiated with the purpose of extending recombination to the 3' portion of the J cluster in the rearranged TCR locus and to the second TCR allele. Dual TCR expression at the cell surface is relatively common in immature thymocytes, but only positively selected TCR is expressed in mature thymocytes (14, 15). The primary (nonselectable) TCR is subject to internalization and intracellular retention (14, 15). Thus, a decision on TCR cell surface expression is made based on TCR engagement during positive selection.

Allelic inclusion of the TCR locus carries the danger of potential reassembly of an autoreactive receptor that may lead to autoimmune reactions (16, 17, 18). A special case of allelic inclusion is termed receptor editing, in which interaction of the primary autoreactive receptor with Ag triggers secondary rearrangement (19). Receptor editing has been first described (20, 21, 22) and subsequently extensively studied in B lymphocytes (reviewed in Ref. 23), and only later found to operate in autoreactive peripheral T cells (24) and thymocytes (18, 25). It is unclear why the risk of autoimmunity is taken by the immune system when safer mechanism(s) of elimination/silencing of autoreactive lymphocytes exists and could be used. In this study, we demonstrate that risk of autoimmunity by TCR editing may be rewarded by more efficient positive selection.

	Materials and Methods

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Experimental animals

C57BL/6 (B6), RAG2^-/-, and H-Y TCR transgenic mice were purchased from Taconic Farms (Germantown, NY). B10.BR (BR), B10.A(2R) (2R), and B10.A(5R) (5R) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). hCD4 transgenic mice on mouse CD4^-/- background were a kind gift from D. R. Littman (New York University School of Medicine). H-Y TCR transgenic B6 mice were bred for two generations with 2R mice to produce the H-Y/2R strain. Littermates were screened by immunofluorescence of peripheral blood cells using (AF6-88.5.3) anti-H-2K^b mAb that does not cross-react to the H-2^k haplotype. To produce H-Y transgenic, RAG2^-/-, hCD4 transgenic B6 strain, we bred B10.D2/H-Y/RAG2^-/- males with hCD4 transgenic females. hCD4-positive F₁ offsprings were intercrossed, and littermates were selected by immunofluorescent staining for both human CD4 (hCD4). Anti-H-2K^b (AF6-88.5.3) and anti-H-2K^d (SF1.1.1) mAbs were used to identify the H-2^d MHC haplotype, anti-hCD4 were used to screen for hCD4 or mCD4, respectively, while anti-B220 Ab was used to determine RAG2 deficiency.

Thymic epithelium grafting

Neonatal B6 or 2R thymi were cultured for 5 days on sponge-supported filters in RPMI medium supplemented with 10% FCS, 50 µM 2-ME, and 1.35 mM deoxyguanosine, as described (26). After 5 days of culture, thymi were placed under the left (B6) or right (2R) kidney capsule of B10.D2/H-Y/RAG2^-/- mice. Mice were allowed to recover and after 4 wk were sacrificed for analysis.

Cell lines and proliferation

Generation and maintenance of the CD4⁺ T cell line from H-Y TCR transgenic mice were described previously (27). T cells (2 x 10⁵) were incubated with irradiated stimulator spleen cells (1 x 10⁶) in round-bottom 96-well plates for 48 h. Each microculture was then pulsed with 0.5 µCi [³H]thymidine for 16 h, and thymidine incorporation was measured on a beta scintillation counter.

Flow cytometry

Ab against the transgenic TCR chain (T3.70) was used as a hybridoma supernatant. Anti-mouse CD4 (H129.19) conjugated to PE, CyChrome-conjugated anti-mouse CD8 (53-6.7), and FITC-conjugated anti-hCD4 were purchased from BD PharMingen (Costa Mesa, CA). Thymocytes were incubated on ice for 30 min with three fluorochrome-conjugated Abs before washing in PBS (2% FCS). Cells were then fixed in 1% paraformaldehyde and analyzed using a FACScan flow cytometer (BD Biosciences, Mountain View, CA). When T3.70 Ab was used, it was followed by multiply adsorbed FITC-conjugated anti-mouse Ig (BD PharMingen).

	Results

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H-Y TCR transgenic T cells recognize H-2A^b in vitro and in vivo

Peripheral CD8⁺ T cells carrying the H-Y-specific transgenic TCR (28) recognize the peptide Smcy_738–746 presented by the H-2D^b MHC class I molecule (29). The same transgenic TCR is also reactive to H-2A^b (27). Although the presence of H-2D^b-associated Ag in the thymus leads to significant deletion of CD4⁺CD8⁺ thymocytes (30, 31), presence of H-2A^b has relatively mild effects (27). Thus, the H-Y TCR transgenic mouse provides a good experimental model to study escape of cells bearing autoreactive TCR. To examine how overt reactivity of the transgenic TCR to H-2A^b might affect the maturation of CD4⁺CD8^- thymocytes, we searched for nonstimulatory MHC class II alleles, so that we could compare CD4⁺CD8^- thymocyte maturation in the neutral and autoreactive backgrounds. Consistent with our previous findings (27), a CD4⁺ cell line isolated from H-Y TCR transgenic mice proliferated upon stimulation with irradiated wild-type B6 (H-2^b), but not upon stimulation with MHC class II-deficient (H-2A^-/-) spleen cells (Fig. 1). C3H/HeJ (H-2^k) spleen cells were also unable to stimulate proliferation, indicating that the TCRs expressed by this cell line do not cross-react with H-2^k class II molecules.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Proliferative response of CD4+ T cells from H-Y TCR transgenic mouse to distinct MHC class II alleles. The HYCD4 cell line was tested for proliferative response to irradiated stimulator cells (1 x 106/well) of wild-type B6, MHC class II-deficient B6, or C3H/HeJ haplotypes. After 2 days of culture, proliferation was determined by tritiated thymidine incorporation. The results represent mean ± SD of triplicate cultures.

View larger version (71K): [in this window] [in a new window]   FIGURE 2. Comparison of H-Y TCR transgenic thymocyte development in B6 or 2R backgrounds. CD4 vs CD8 plots of thymocytes isolated from male (A) or female (B) H-Y TCR transgenic mice.

Reduced numbers of CD4⁺CD8⁺ thymocytes could result from physical deletion, or by clonal arrest (32). Apoptosis of CD4⁺CD8⁺ H-Y TCR transgenic thymocytes could be readily induced in suspension cultures by H-2D^b-presented male Ag, but not by self H-2A^b autoantigen (data not shown), arguing against the physical deletion. Development of H-Y thymocytes is slower in the presence of H-2A^b relative to the MHC class II-deficient background (33), indicating that indeed, H-2A^b may induce clonal arrest. To address this issue directly, female B10.D2/H-Y/RAG2^-/- mice were grafted with female B6 and 2R thymic epithelium. Four weeks later, there were twice as many CD4^-CD8^- thymocytes in the B6 grafts than in the 2R grafts of the same hosts (Fig. 3), indicating temporal arrest in thymic development. In addition, there was 3- to 4-fold less CD4^-CD8⁺ thymocytes in B6 grafts, most likely reflecting a cumulative effect of fewer CD4⁺CD8⁺ cells auditioning for selection due to the arrest at an earler stage. Some CD4⁺CD8^- thymocytes were present in both B6 and 2R grafts, but they were not fully mature as they expressed low levels of CD8 molecules (for example, mean CD8 fluorescence of CD4⁺CD8^- cells was 52 compared with 26 in CD4^-CD8^- thymocytes). Thus, our data suggest that developmental arrest appears to be the only mechanism of H-2A^b-induced negative selection.

View larger version (31K): [in this window] [in a new window]   FIGURE 3. Arrested development of CD4+CD8+ H-Y TCR transgenic thymocytes in B6 thymic epithelium grafts. B10.D2/RAG2-/-/H-Y TCR transgenic females were grafted with dGuo-treated B6 or 2R neonatal thymi. Same animals received both grafts under different kidney capsules. Four weeks after grafting, thymocytes were isolated from the grafts and analyzed for CD4 and CD8 expression by flow cytometry.

Down-modulation of transgenic TCR chains in CD4⁺CD8^- thymocytes

One of the most immediate consequences of positive selection is up-regulation of cell surface TCR levels. When thymocytes express two TCRs, the up-regulation is limited to the TCR that promoted positive selection (14). Therefore, up-regulation of a TCR is an indicator of the TCR used to promote positive selection. In the H-Y TCR transgenic mice, CD4^-CD8⁺ thymocytes express relatively high levels of transgenic TCR chain. In contrast, few CD4⁺CD8^- thymocytes express detectable cell surface transgenic TCR (11). This fact, together with the presence of CD4^-CD8⁺, and absence of CD4⁺CD8^- thymocytes in rearrangement-deficient backgrounds (34), indicates that positive selection of these thymocyte subsets is mediated by transgenic or endogenous TCR chains, respectively.

To determine which TCR is used for selection of CD4⁺CD8^- thymocytes in 2R background, we compared the levels of transgenic TCR expressed by this thymocyte subset in 2R vs B6 backgrounds. The pattern of transgenic TCR expression in CD4⁺CD8^- vs CD4^-CD8⁺ thymocytes in 2R and B6 backgrounds is virtually identical (Fig. 4), suggesting that endogenous TCR rearrangements allow selection of CD4⁺CD8^- thymocytes in the 2R background as well. The expression of transgenic TCR chain was high in all thymocyte subsets in both genetic backgrounds (data not shown), indicating that allelic exclusion at the TCR locus was not affected by H-2A^b.

View larger version (14K): [in this window] [in a new window]   FIGURE 4. Cell surface expression of transgenic TCR on thymocyte subsets from female B6 or 2R H-Y TCR transgenic mice. Thymocytes isolated from female H-Y TCR transgenic mice of either B6 or 2R background were labeled with anti-CD4, anti-CD8, and T3.70 mAbs and analyzed by flow cytometry. The histograms represent comparison of transgenic TCR expression on gated CD4+CD8+, CD4-CD8+, or CD4+CD8- thymocyte populations from the two backgrounds.

Selection of CD4⁺CD8^- thymocytes by endogenous TCR chains in both B6 and 2R backgrounds and reactivity of the H-Y transgenic TCR to H-2A^b present a unique opportunity to directly compare maturation of CD4⁺CD8^- thymocytes under conditions of allelic inclusion (2R background) with those of replacing the autoreactive TCR (B6 background). Three breeding strategies were used to compare the numbers of CD4⁺CD8^- thymocytes in B6 and 2R backgrounds. To our surprise, the numbers of CD4⁺CD8^- thymocytes were reduced in neutral 2R background, relative to the autoantigenic B6 background (Fig. 2B; Table I). This was not due to the influence of genetic backgrounds, as the numbers of CD4⁺CD8^- thymocytes in TCR nontransgenic B6 or 2R mice are similar (Table I). The presence of a single H-2A^b allele was sufficient to significantly increase selection of CD4⁺CD8^- thymocytes in the 2R background (Table I, breeding strategy 3). The difference between the 2R and B6 background was also evident when absolute numbers of CD4⁺CD8^- thymocytes were calculated (Fig. 5). Thus, H-2A^b induces more efficient positive selection of H-Y TCR transgenic CD4⁺CD8^- thymocytes than H-2A^k and H-2E^k combined.

View larger version (33K): [in this window] [in a new window]   FIGURE 5. Absolute number of CD4-CD8+ or CD4+CD8- H-Y TCR transgenic thymocytes in the absence or presence of H-2Ab. Percentage of CD4-CD8+ or CD4+CD8- thymocytes from female F1(2R x D2) or F1(B6 x D2) offsprings (see Table I) was multiplied by individual total thymus cell numbers. Shown are mean and SEs.

hCD4 expression enhances endogenous TCR-mediated selection of CD4⁺CD8^- H-Y TCR transgenic thymocytes

In the absence of endogenous TCR rearrangements, no CD4⁺CD8^- H-Y TCR transgenic thymocytes are selected (34), suggesting that the affinity/avidity of the transgenic TCR is either below or above the threshold required for positive selection. To address this question, we have determined the effect of hCD4 coreceptor expression (35). hCD4 interacts functionally with mouse MHC class II equally well as the mouse CD4 (36). We reasoned that bringing extra p56^lck molecules into the TCR recognition cluster via additional CD4 should lead to a stronger signal. Although the expression of mouse CD4 is somewhat reduced in hCD4 transgenic mice, the overall CD4 levels (mouse plus human) are functionally higher than the levels of mouse CD4 in wild-type mice. This is suggested by more pronounced reduction of H-Y TCR transgenic CD4⁺CD8⁺ thymocytes in male mice expressing the hCD4 (Fig. 6A). Identical results were obtained with mice bred to the RAG2^-/- background (Fig. 6B).

View larger version (52K): [in this window] [in a new window]   FIGURE 6. The effects of hCD4 transgene expression on development of H-Y TCR transgenic thymocytes in the B6 background. Flow cytometry analysis of thymocytes isolated from male (A and B) or female (C and D) RAG2+/+ (A and C) or RAG2-/- (B and D) H-Y TCR transgenic mice. Shown are CD4 vs CD8 plots of wild-type vs hCD4 backgrounds.

	Discussion

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The H-Y transgenic TCR reacts with H-2A^b sufficiently strongly to induce proliferation and/or cytokine secretion of peripheral CD4⁺ T cells (27). The autoantigen is present in the thymus, and thymocytes interact with it before positive selection. This interaction, however, does not result in positive selection, as evidenced by absence of CD4⁺CD8^- thymocytes in RAG-2-deficient background. Therefore, the signal delivered by H-2A^b is either too weak, or too strong for positive selection. Given that signal strength delivered by the same Ag is sufficient to induce proliferation of peripheral T cells, and that increasing the selecting signal strength via hCD4 expression failed to rescue CD4⁺CD8⁺ thymocytes when endogenous rearrangements were prevented, we believe that the latter possibility (signal too strong) is a more likely explanation for absence of positive selection by the H-2A^b. One of the characteristics of receptor editing is RAG-1/2 re-expression in the cell population interacting with the autoantigen. However, CD4⁺CD8⁺ thymocytes that encounter H-2A^b already express high levels of RAG-1/2 as part of the regular developmental program (5). Thus, direct demonstration of this aspect of receptor editing in this model is not possible. However, requirement for endogenous TCR for CD4⁺CD8^- thymocyte selection in B6 background, reactivity of the transgenic TCR for H-2A^b, and absence of CD4⁺CD8^- thymocyte selection in RAG-2^-/-/hCD4 transgenic background collectively argue that endogenous TCR rearrangement is due to receptor editing.

Replacement of the transgenic TCR by endogenously rearranged TCR chains occurred in both B6 and 2R CD4⁺CD8^- thymocytes. However, in the B6 background, in which CD4⁺CD8⁺ thymocytes in addition encounter the autoantigen, CD4⁺CD8^- thymocytes are more abundant than in a neutral (2R) environment (Table I), suggesting that receptor editing results in relatively more efficient positive selection. What is the possible mechanism of efficient positive selection? The most likely explanation is that TCR itself contributes substantial avidity for H-2A^b, reaching almost the threshold required for positive selection (Fig. 7). Consequently, a wide range of endogenous TCR chains can raise the total avidity of the TCR to fit between the thresholds required for positive and negative selection. The transgenic TCR and occasional endogenous TCR chains can raise the avidity of the TCR over the threshold for negative selection. In the case of H-2A^k/H-2E^k class II molecules, only a narrow range of endogenous TCR chains can contribute to the low avidity of transgenic TCR to rescue selection. Presence of the hCD4 lowers thresholds for both positive and negative selection. In this case, the number of TCRs transferred from nonselectable to the selectable range of avidities is greater than the number of selectable TCRs eliminated because they entered the range of avidities sufficient for negative selection. Hence, the net result of lowering both thresholds is a gain in the number of selected TCRs.

View larger version (35K): [in this window] [in a new window]   FIGURE 7. Model that explains relative efficiencies of TCR repertoire selection by receptor editing vs allelic inclusion. See text for detailed explanation.

In 1959, Burnet (40) proposed a clonal selection theory that attempted to explain the specificity of induction of immune responses, and mechanisms of nonresponsiveness to self. A central tenet of this widely accepted theory was that lymphocytes may express only one Ag receptor to assure the specificity of the immune response strictly to Ags. Expression of two receptors was postulated to be especially dangerous if one of the receptors was autoreactive, because activation through nonautoreactive receptor could lead to the expansion of autoreactive cells. The phenomenon of repressing expression of Ag receptor encoded by the second allele was named allelic exclusion. Although the clonal selection theory was shown to be correct in many aspects, several exceptions to the concept of "one lymphocyte-one receptor" have been described, obliging modifications of the original hypothesis to account for these observations. Even though allelic exclusion was shown to operate at the Ig H chain and the TCR loci, thanks to the inclusion of the Ig L chains and the TCR loci, a portion of both B and T lymphocytes expresses two Ag receptors (reviewed in Ref. 23). That observation prompted testing Burnett’s hypothesis that lymphocytes carrying two receptors would be capable of triggering autoimmunity. Indeed, autoreactive TCRs could escape negative selection because of poor autoantigen presentation (16, 18), or because of inadequate signal strength due to low density of the autoreactive receptor (17). Thus, the immune system appears to risk development of autoimmunity for the benefit of efficient positive selection.

T cells responsive to autoantigens implicated in autoimmune disorders can be isolated from healthy individuals (41). It is therefore important to distinguish autoreactivity (reactivity of immune receptors with components of self) from autoimmunity (destructive immune response directed at cells expressing components of self). Different checkpoints need to be passed to develop autoimmunity (42). The mechanisms that normally prevent autoreactive lymphocytes to become autodestructive include, but are not limited to: regulatory cells, tissue-specific expression and balance between expression of activating and inhibitory costimulatory molecules, secretion of immunomodulatory cytokines (such as TGF or IL-10), and Fas-Fas ligand interaction. Because of these multiple checkpoints that prevent the development of autoimmunity, and a significant benefit of efficient TCR repertoire selection, editing autoreactive TCRs must be considered safe relative to the advantages it confers to the host.

	Acknowledgments

 
We thank John Hirst for FACS analysis, Dan Littman for providing hCD4 transgenic mouse strain, Nigel Kileen for helpful discussions, and Alan Frey for reading the manuscript.

	Footnotes

 
¹ This work was supported by the National Institutes of Health Grant AI41573 (to S.V.), and National Cancer Institute Core Support Grant 5P30 CA16087.

² Current address: Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, NY 10032.

³ Address correspondence and reprint requests to Dr. Stanislav Vukmanovi, Michael Heidelberger Division of Immunology, Department of Pathology, New York University School of Medicine, 550 First Avenue, New York, NY 10016. E-mail address: vukmas01{at}med.nyu.edu

⁴ Abbreviations used in this paper: RAG, recombination-activating gene; hCD4, human CD4.

Received for publication September 27, 2001. Accepted for publication June 6, 2002.

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