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The Journal of Immunology, 2000, 165: 3073-3079.
Copyright © 00 by The American Association of Immunologists

Thymic Selection Generates T Cells Expressing Self-Reactive TCRs in the Absence of CD451

Sébastien Trop*, Josée Charron{dagger}, Chantal Arguin{dagger} and Patrice Hugo2,*,{dagger}

* Division of Experimental Medicine, Department of Medicine, McGill University, Montréal, Québec, Canada; and {dagger} PROCREA BioSciences, Montréal, Québec, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The CD45 protein tyrosine phosphatase regulates Ag receptor signaling in T and B cells. In the absence of CD45, TCR coupling to downstream signaling cascades is profoundly reduced. Moreover, in CD45-null mice, the maturation of CD4+CD8+ thymocytes into CD4+CD8- or CD4-CD8+ thymocytes is severely impaired. These findings suggest that thymic selection may not proceed normally in CD45-null mice, and may be biased in favor of thymocytes expressing TCRs with strong reactivity toward self-MHC-peptide ligands to compensate for debilitated TCR signaling. To test this possibility, we purified peripheral T cells from CD45-null mice and fused them with the BW{alpha}-ß- thymoma to generate hybridomas expressing normal levels of TCR and CD45. The reactivity of these hybridomas to self or foreign MHC-peptide complexes was assessed by measuring the amount of IL-2 secreted upon stimulation with syngeneic or allogeneic splenocytes. A very high proportion (55%) of the hybridomas tested reacted against syngeneic APCs, indicating that the majority of T cells in CD45-null mice express TCRs with high avidity for self-MHC-peptide ligands, and are thus potentially autoreactive. Furthermore, a large proportion of TCRs selected in CD45-null mice (H-2b) were also shown to display reactivity toward closely related MHC-peptide complexes, such as H-2bm12. These results support the notion that modulating the strength of TCR-mediated signals can alter the outcome of thymic selection, and demonstrate that CD45, by molding the window of affinity/avidity for positive and negative selection, directly participates in the shaping of the T cell repertoire.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Signals mediated by the pre-TCR and TCR are crucial for the development of {alpha}ß T cells (reviewed in Refs. 1, 2, 3). At the CD4-CD8- double-negative stage of development, signals delivered through the pre-TCR allow immature T cells to proliferate and differentiate into CD4+CD8+ double-positive (DP)3 thymocytes. At this stage, the pre-TCR is replaced by TCR-{alpha}ß heterodimers. Thymocytes that express a TCR with too low affinity or avidity for self-MHC-peptide ligands expressed on thymic stromal cells undergo programmed cell death (death by neglect), as do thymocytes that express a TCR with too high affinity or avidity for their ligand (negative selection). Only thymocytes expressing a TCR with intermediate reactivity toward self-MHC-peptide complexes are allowed to mature to the CD4+CD8- or CD4-CD8+ single-positive (SP) stage (positive selection), and subsequently migrate to the periphery (4, 5). These findings have been incorporated into a signal strength model of selection, which views positive and negative selection as distinct programmed responses with different activation thresholds, positive selection having a lower threshold of activation than negative selection (6, 7, 8, 9, 10). The window of affinity/avidity allowing positive but not negative selection is thus plastic, not static, as it depends on the strength of the signals delivered through the TCR.

TCR signaling may be attenuated in several of the following ways: by decreasing the amount of ligand on the APC or thymic stromal cell (11, 12, 13); by lowering the density of TCR at the surface of the T cell (14); by preventing the relocalization of activated TCRs to the detergent-resistant lipid microdomains of the plasma membrane, which contain elements of the TCR signaling machinery (15); or by altering the quantity or enzymatic activity of signaling molecules recruited by the TCR, such as the Lck protein tyrosine kinase (PTK; Ref. 16). According to the signal strength model of TCR selection, blunting TCR signaling by any of these methods should hinder the positive selection of thymocytes expressing TCRs with intermediate affinity/avidity for self-MHC-peptide ligands, and permit thymocytes expressing weakly autoreactive TCRs to escape negative selection and be positively selected. Thymocytes bearing strongly autoreactive TCRs would still receive sufficiently strong signals to be eliminated through apoptosis.

In support of this notion, analysis of mice lacking various components of the TCR signaling complex has revealed that deficient signaling through the TCR can affect the outcome of thymic selection. For instance, it has been shown using a TCR transgenic mouse model that decreasing the density of TCR at the cell surface can impair negative selection, and that this defect can be compensated by increasing the amount of MHC-peptide ligands recognized by the TCR (14). It has also been reported that in the absence of CD3-{zeta} and CD3-{eta}, which are required for normal surface expression of the TCR and amplification of TCR-mediated signals, negative selection is impaired such that T cells accumulate in the periphery that react with self-MHC upon restoration of normal surface levels of TCR (17).

The CD45 protein tyrosine phosphatase is abundantly expressed in lymphocytes, and is required for efficient signaling through the TCR and the B cell (Ig) receptor (18). Accordingly, signaling through the pre-TCR and TCR is hampered in CD45-deficient thymocytes, affecting both the transition from the double-negative stage to the DP stage, and from the DP stage to the SP stage (19, 20). Consequently, in CD45-null mice, the absolute number of DP thymocytes is reduced by about 2-fold compared with wild-type littermates, and the number of SP thymocytes is reduced by a further 5-fold (20), underscoring the importance of CD45 in T cell development. In vitro studies have shown that CD45 can dephosphorylate several substrates involved in TCR signaling, including the Src-family PTKs, Lck and Fyn, as well as CD3-{zeta} and the ZAP-70 PTK (21, 22, 23, 24). The ability of CD45 to regulate the activity of Src-family PTKs, which are required for the initiation of the signal-transduction cascades associated with the TCR (25), suggests a key role for CD45 in modulating TCR-mediated signals. Indeed, studies using thymocytes isolated from CD45-null mice have shown that CD45 is responsible for dephosphorylating the negative regulatory tyrosine residue in the carboxyl terminus of Lck (Y505) and Fyn (Y531) (26). This causes an increase in the activity of these kinases by freeing their SH2 domain to interact with other signaling molecules (27, 28). However, the regulation of Src-family kinases by CD45 in vivo appears complex, as recent reports suggest that CD45 may reduce rather than enhance the activity of these enzymes, due to dephosphorylation of the positive regulatory tyrosine within the kinase domain (29, 30). Regardless of the manner in which CD45 regulates the activity of Src-family PTKs, T cells from CD45-null mice exhibit profoundly impaired activation and development that can be rescued by the introduction of a transgene encoding constitutively active Lck (LckY505F; Refs. 31 and 32). Hence, CD45 is generally viewed as a positive regulator of Ag receptor signaling in T and B cells.

Therefore, according to the signal strength model of thymic selection, the T cell repertoire in CD45-null mice should be skewed toward increased affinity/avidity for self-MHC-peptide Ags to compensate for the weaker signals generated upon engagement of the TCR. Thus, a stronger interaction of the TCR with its ligand may be required to achieve positive selection in the absence of CD45, thereby producing potentially self-reactive mature T cells that would otherwise have been deleted. However, because peripheral T cells in CD45-null mice are severely impaired in their ability to respond to TCR ligation (19, 20, 26), they are nevertheless tolerant to self. Therefore, we hypothesized that complementation of peripheral T cells from these mice with normal levels of CD45 should unmask their likely autoreactive potential. To test this hypothesis, we restored surface expression of CD45 to T cells from CD45-null mice by fusing them with the BW{alpha}-ß- thymoma, thereby generating T cell hybridomas expressing CD45. We then analyzed the reactivity of these hybridomas toward self-MHC-peptide Ags. We observed that a very high proportion of these cells were reactive to self-MHC-peptide ligands. Our results confirm and extend earlier findings that CD45 can alter thymic selection (33, 34, 35, 36, 37) by providing the first evidence that negative selection of an endogenous repertoire of TCRs by self-MHC-peptide ligands is impaired in the absence of CD45. These results support the notion that modulating the strength of TCR-mediated signals can alter the outcome of thymic selection, and demonstrate that CD45, by molding the window of affinity/avidity for positive and negative selection, plays a key role in the shaping of the T cell repertoire.


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

The CD45 exon 9-deficient mice have been described (20, 26). These mice were originally bred on the C57BL/6 background for six generations and carry the H-2b haplotype. C57BL/6 (H-2b), C57BL/10 (H-2b), B6.C-H2bm12 (H-2bm12), DBA/2 (H-2d), and B10.BR (H-2k) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in a specific pathogen-free animal facility at the Institut de Recherches Cliniques de Montréal (Montréal, Canada).

Hybridomas

The BW5147 TCR{alpha}-ß- thymoma cell line lacks CD3-{delta}, TCR-{zeta}{eta} and FcR{gamma} transcripts, as well as functional TCR-{alpha} and ß-chain genes (38, 39, 40, 41). To generate hybridomas from T cells lacking CD45, lymph node T cells from CD45 exon 9-deficient mice were purified by passage through a nylon wool column (42) and fused to BW5147 TCR-{alpha}-ß- thymoma cells (43). After hypoxanthine/aminopterin/thymidine selection, the resulting T cells hybridomas, termed 45E9T, were analyzed by staining for cell surface expression of TCR-ß-chain and CD45. Hybridomas expressing both markers were cloned and further characterized as described below. Lymph node T cells from wild-type (CD45+/+) C57BL/6 mice were similarly purified and fused to BW5147 TCR-{alpha}-ß- thymoma cells to produce B6THyb hybridomas. The AD10Thyb9.7 T cell hybridoma has been described (44). The KSEA-1.8, KS-20.15, KMls-12.6, 5KC-73.8, 4B-1810, B4V4D8.22, 3B2-10.4, and AODH-3.4 T cell hybridomas (38, 43, 45) were kindly provided by P. Marrack and J. Kappler (Howard Hughes Medical Institute, Denver, CO). The KSEA-1.8, KS-20.15, KMls-12.6, and 5KC-73.8 hybridomas were derived from the B10.BR mouse strain (H-2k), and had BW{alpha}-ß- as a fusion partner. The 4B-1810, B4V4D8.22, and 3B2-10.4 hybridomas were obtained by fusing Con A blasts from C57BL/10 mice (H-2b) to BW{alpha}-ß-. The AODH-3.4 hybridoma expresses TCR specific for keyhole limpet hemocyanin presented by I-Ak and alloreactive to I-Ab, and was obtained by fusing T cells from DBA/2 mice (H-2d) to AO-40.10 thymoma cells, which were derived from the AKR mouse strain (H-2k); the TCR restricted onto H-2d was lost in the fusion process. The 1BVB11-17.7 T cell hybridoma (46) obtained by fusing T cells from C57BL/6 mice (H-2b) to BW{alpha}-ß- was a kind gift of E. Palmer (Basel Institute, Basel, Switzerland). All cell lines used in this study were cultured in complete RPMI 1640 medium (Mediatech, Herndon, VA) supplemented with 10% FCS (BioMedia, Kirkland, Quebec, Canada).

Abs and flow cytometric analysis

The level of TCR and CD45 expression on T cell hybridomas was determined by staining 106 cells with saturating concentrations of FITC-conjugated ALI 4A2 (anti-CD45) mAb (47) and Red670-conjugated H57-597 (anti-TCRß) mAb (Ref. 48 ; Life Technologies, Rockville, MD) for 25 min at 4°C in PBS containing 2% FCS and 0.1% sodium azide. To determine Vß expression on T cell hybridomas, 106 cells were incubated with unconjugated Vß-specific mAbs, followed by staining with FITC-conjugated 187.1 (anti-mouse IgG2a; Ref. 49) mAb or MAR18.5 (anti-rat IgG2a; Ref. 50) mAb, as above. Culture supernatants from the following clones were prepared in our laboratory: B20.6 (Vß2; Ref. 51), MR9-8 (Vß5.1; Ref. 52), KJ16-133 (Vß8.1 and Vß8.2; Ref. 53), F23.2 (Vß8.2; Ref. 54), RR3-15 (Vß11; Ref. 55), 14.2 (Vß14; Ref. 56). Gated events (2 x 104) were acquired on a Coulter EPICS XL (Coulter Electronics, Montréal, Quebec, Canada), and analyzed with the CellQuest software (Becton Dickinson, San Jose, CA).

Stimulation of T cell hybridomas

In brief, splenocytes were treated with 25 µg/ml mitomycin C (Sigma-Aldrich, Oakville, Ontario, Canada) for 20 min at 37°C, followed by four washes in complete RPMI 1640 medium. Microcultures were then prepared by mixing 105 responding cells and 106 splenocytes in a final volume of 200 µl. For stimulation with the anti-TCRß mAb H57-597, flat-bottom Pro-Bind microtiter plates (Becton Dickinson, Franklin Lakes, NJ) were precoated with 40 µl of a 5 µg/ml solution of Ab. Cultures were incubated for 24 h, at which time culture supernatants were harvested and assayed for the level of IL-2 using the IL-2-dependent cell line HT-2 (57). After 16 h, 1 µCi/well of [3H]thymidine (DuPont NEN, Boston, MA) was added to the HT-2 cultures, and cells were incubated for a further 8 h. The amount of [3H]thymidine incorporated into HT-2 cells was determined by transferring cell lysates onto glass fiber filtermats (Wallac, Turku, Finland), followed by scintillation counting on a Betaplate counter (Wallac).

Statistical analysis

Data are presented as mean ± SD values, and the statistical significance of differences between groups was determined by Student’s unpaired t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fusion of peripheral SP T cells from CD45-null mice with BW{alpha}-ß- generates hybridomas that express normal levels of TCR and CD45

To study the reactivity of mature T cells emerging from thymic selection in CD45-null mice, we first restored normal levels of CD45 expression to these cells by fusing them to the BW{alpha}-ß- thymoma. BW{alpha}-ß- is a variant of the BW5147 thymoma that lacks functional TCR-{alpha} and -ß genes, and has been used in the past to analyze the specificity of TCRs expressed by heterogeneous populations of T cells (11, 58).

Once produced, the T cell hybridomas, termed 45E9T, were expanded and analyzed for surface expression of TCR and CD45. Results obtained with six representative 45E9T hybridomas are shown in Fig. 1Go. As expected, BW{alpha}-ß- thymoma cells lacked expression of TCR, but showed high levels of CD45 at their surface. The 45E9T.29 hybridoma, which did not express TCR at its surface, was used as negative control in subsequent experiments. Of the 29 hybridomas analyzed initially, 19 (65%) expressed detectable levels of TCR at their surface. The number of TCR-expressing hybridomas was subsequently reduced to 11, most likely due to loss of one or more genes encoding the TCR-{alpha} or -ß-chains, or the TCR-{zeta} subunit, which is required for efficient TCR expression (59, 60, 61) and is lacking in the BW{alpha}-ß- thymoma. In fact, it proved necessary in some cases to sort 45E9T hybridomas for TCR expression, as cell populations that were originally homogeneous gave rise on occasion to mixed populations containing both TCR+ and TCR- hybridomas.



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  		FIGURE 1. 45E9T hybridomas express normal levels of TCRß and CD45 at their surface. Peripheral SP T cells isolated from the lymph nodes of CD45 exon 9 knock-out mice were fused with the BW{alpha}-ß- thymoma. The resulting hybridomas, termed 45E9T, were analyzed by flow cytometry after staining with anti-TCRß and anti-CD45 mAb. Unstained cells (Control) were used to establish baseline fluorescence levels, and the AD10THyb9.7 T cell hybridoma was included as positive control. The 45E9T.29 hybridoma, which does not express TCR, was used as a negative control in subsequent experiments.

 
Most 45E9T hybridomas are specific for self-MHC-peptide complexes

Before examining the specificity of the 45E9T hybridomas, we first tested their ability to secrete IL-2 upon stimulation with immobilized anti-TCRß mAb. Results obtained with the same six representative hybridomas described in Fig. 1Go showed that the level of IL-2 secretion was comparable between each clone (Fig. 2Go, top left). Similar results were also observed with the other hybridomas (data not shown). As expected, no IL-2 secretion could be detected with the TCR- 45E9T.29 hybridoma.



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  		FIGURE 2. Reactivity of 45E9T hybridomas against self or foreign MHC-peptide ligands. IL-2 production of 45E9T hybridomas (105 cells/well) was measured after a 24-h incubation period in wells coated with 5 µg/ml anti-TCRß mAb or with splenocytes (106 cells/well) from various mouse strains, as described in Materials and Methods. CD45-/- and C57BL/6 mice carry the H-2b haplotype; B6.C-H2bm12 mice carry the H-2bm12 haplotype, which contains a mutated I-Ab allele (62 ); and DBA/2 and B10.BR mice carry the H-2d and H-2k haplotypes, respectively. The 45E9T.29 hybridoma was used as negative control. Mean values ± SD for triplicate samples are given. Values were corrected for spontaneous IL-2 secretion. The asterisks indicate responses at least 2-fold greater relative to background levels and of statistical significance (Student’s t test; p < 0.01). The results are representative of at least three independent experiments.

 
We next assessed the response of the 45E9T hybridomas to syngeneic spleen cells (i.e., from CD45 exon 9 knock-out mice) expressing self-MHC-peptide ligands, and to spleen cells from allogeneic mouse strains. CD45-null mice were backcrossed onto the C57BL/6 (H-2b) background for only six generations, and thus contain residual DNA from the (129/Sv X 129/Sv-CP)F1 embryonic stem cells used to obtain germline transmission of the CD45 mutation (20). Thus, APCs derived from C57BL/6 mice express the same MHC molecules as syngeneic APCs, but may present a slightly different library of peptides, owing to differences in genotype. The B6.C-H2bm12 congenic strain was derived from the C57BL/6 strain, and carries point mutations resulting in three amino acid changes in the I-Aß-chain (62), all of which have been shown to affect TCR interaction (63), presumably by altering the epitope repertoire presented by I-A molecules (64). Hence, a fraction of the class II MHC-peptide complexes expressed in these mice are foreign with respect to the H-2b background. DBA/2 and B10.BR mice carry the H-2d and H-2k haplotypes, respectively, the former being more closely related to H-2b than the latter (65). When stimulated with syngeneic APCs (CD45-/- H-2b), several 45E9T hybridomas displayed anti-self reactivity (Fig. 2Go, top right). Because the strength of the response varied significantly between clones, we defined autoreactivity as a response that resulted in at least a 2-fold increase in IL-2 secretion over background levels; this scheme was also used to score alloreactivity. Thus, clones 15/1, 17, and 24/1 were considered autoreactive, whereas clones 8/1 and 9/2 were not (Fig. 2Go, top right). A similar pattern of reactivity was also observed with APCs from C57BL/6 mice (Fig. 2Go, middle left). However, responses were weaker, such that only clones 17 and 24/1 scored positive for alloreactivity. As discussed above, this may be explained by the slight difference in the nature of the MHC-peptide complexes presented at the surface of APCs from C57BL/6 mice, compared with those from CD45-null mice. When 45E9T hybridomas were stimulated with APCs from B6.C-H2bm12 mice, clone 17 but not clone 24/1 yielded a positive response (Fig. 2Go, middle right), which was somewhat weaker than that obtained with C57BL/6 APCs. This might be a consequence of the ability of I-Abm12 molecules to present different peptide epitopes compared with wild-type I-A molecules (64). Stimulation with spleen cells from DBA/2 mice (H-2d), expressing more distantly related MHC ligands, triggered clones 8/1 and 17 to secrete high amounts of IL-2 (Fig. 2Go, bottom left). None of the 45E9T hybridomas tested displayed alloreactivity to H-2k (Fig. 2Go, bottom right), which reflects the considerable disparity between H-2k and H-2b (65).

Data obtained with all eleven 45E9T hybridomas tested are summarized in Fig. 3GoA. In total, 8/11 (73%) clones displayed auto and/or alloreactivity. Autoreactivity was observed with 6/11 (55%) clones. Reactivity to C57BL/6, B6.C-H2bm12, and DBA/2 was distributed equally, occurring with 3–4/11 (27–36%) of the clones tested. Again, none of the 45E9T hybridomas reacted against APCs from B10.BR mice. To eliminate the possibility that autoreactive hybridomas might express a common TCR, each clone was stained with a panel of Vß-specific mAbs and analyzed by flow cytometry. Each of the six self-reactive clones was found to express a different TCR-ß-chain (Table IGo). Together, these results indicate that the T cell repertoire selected in CD45-null mice is strongly biased toward recognition of self-MHC-peptide complexes, or closely related ligands. To ensure that the self-reactivity noted for a large proportion of 45E9T hybridomas in this study did not result from the fusion process with a cell partner, we generated a second set of hybridomas, B6THyb, by fusing peripheral T cells from wild-type C57BL/6 mice with BW{alpha}-ß- thymoma cells. B6THyb that expressed TCR secreted IL-2 upon stimulation with immobilized anti-TCRß mAb (data not shown). In contrast to the results obtained with 45E9T hybridomas, none of the B6THyb hybridomas tested displayed autoreactivity, and only 1/15 (7%) reacted against allogeneic (DBA-2) APCs (Fig. 3GoB). Furthermore, we also tested the auto and alloreactive potential of various hybridomas produced by other investigators, derived from T cells restricted either to the H-2b or H-2k haplotype, and fused to the BW{alpha}-ß- fusion partner, as were the 45E9T hybridomas. These hybridomas expressed TCR at their surface, and could secrete IL-2 upon stimulation with anti-TCRß mAb (data not shown). Significantly, none of these hybridomas displayed self reactivity (Fig. 4Go). Moreover, only one hybridoma, KMls-12.6, was observed to react to allogeneic APCs. The T cell hybridoma AODH-3.4 was used as a positive control for the detection of alloreactive responses. This hybridoma, which has the AO-40.10 thymoma as a fusion partner, has been shown to be reactive to H-2b and H-2d, but tolerant to H-2k (Fig. 4Go; Ref. 43).



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  		FIGURE 3. Most 45E9T hybridomas display auto or alloreactivity. A, 45E9T hybridomas were stimulated with splenocytes from various mouse strains, including autologous splenocytes from CD45-null mice, and IL-2 production was assayed as described in Materials and Methods. B, B6THyb hybridomas were similarly stimulated with autologous or allogeneic splenocytes. Responses resulting in at least a 2-fold increase in IL-2 secretion over background levels were considered positive, and are indicated by dark squares. The experiments were repeated at least four times, with similar results.

 

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  		Table I. Self-reactive 45E9T hybridomas each express a different TCR-ß-chain

 


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  		FIGURE 4. Hybridomas derived from wild-type mice do not display autoreactivity. Hybridomas derived from C57BL/6, C57BL/10, or B10.BR mice were tested for anti-self reactivity and alloreactivity (see Materials and Methods). All hybridomas were produced with BW{alpha}-ß- as a fusion partner, except AODH-3.4, which has AO-40.10 as a fusion partner. Responses resulting in at least a 2-fold increase in IL-2 secretion over background levels were considered positive, and are indicated by dark squares. Results are representative of three independent experiments.

 
Together, these experiments demonstrate that auto and alloreactivity are seldom observed with T cell hybridomas derived from CD45+/+ mice, and highlight the strong bias toward autoreactivity that results from anomalous thymic selection in CD45-deficient mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study addresses the possibility that the protein tyrosine phosphatase CD45 influences the outcome of thymic selection and directly participates in the shaping of the T cell repertoire. Specifically, we sought evidence that the aberrant TCR-mediated signaling occurring in the absence of CD45 could result in impaired negative selection of thymocytes expressing TCRs with high reactivity toward self-MHC-peptide ligands, and thus lead to the maturation of potentially autoreactive T cells. Indeed, our results strongly suggest that in CD45-deficient mice, the majority (over 50%) of peripheral T cells express TCRs with high affinity for self-MHC-peptide Ag. Moreover, a large proportion of the TCRs positively selected on H-2b molecules in CD45-null mice were also observed to display reactivity toward closely related MHC-peptide complexes, such as those presented in B6.C-H-2bm12 mice.

Previous experiments aimed at defining the role of CD45 in thymic selection have generated contradictory results. Ong et al.(33) reported that introduction of a CD45 transgene in male H-Y TCR transgenic mice, which increased CD45 expression, led to enhanced negative selection of DP thymocytes, and that positive selection of CD8+ SP thymocytes was increased in H-Y TCR transgenic female mice (35), consistent with a positive role for CD45 in signaling through the TCR. In keeping with these results, Conroy et al. (34) later showed that negative selection of TCR Vß8+ thymocytes in response to the superantigen staphylococcal enterotoxin B was impaired in fetal thymic organ cultures from CD45-null embryos. More recently, Mee et al. (37) demonstrated that both positive and negative selection of a transgenic TCR are impaired in the absence of CD45. Evidence that CD45 may also function to attenuate TCR signaling has also been elicited using a similar experimental approach. In a study employing mice expressing lower levels of CD45 owing to the presence of only one functional copy of the CD45 gene, and transgenic for the P14 TCR specific for the lymphocytic choriomeningitis virus, Wallace et al. (36) showed that positive selection could be enhanced by a decrease in CD45 expression. Moreover, this study also reported that reduced expression of CD45 rendered P14 TCR+ thymocytes susceptible to negative selection by a variant of lymphocytic choriomeningitis virus that is under normal conditions inefficient at inducing negative selection, suggesting that decreased CD45 expression also lowered the threshold for negative selection. It has been argued that the results obtained in the latter study reflect an increase in the avidity of the interaction between transgenic thymocytes and intrathymic ligands in CD45+/- mice, due to reduced steric hindrance by CD45 molecules, and also to increased TCR levels at the cell surface (36). It is possible that CD45 may influence the selection of individual TCRs in either a positive or negative manner, as has been shown for CD5, another transmembrane protein expressed at the surface of T cells (66).

Such dichotomy in the effect of CD45 over or underexpression on thymic selection reflects the complex regulatory function of CD45 in vivo. CD45 is generally viewed as a positive regulator of TCR signaling owing to its ability to dephosphorylate the negative regulatory tyrosine residue of Src-family PTKs, such as Lck and Fyn, thereby enhancing the activity of these kinases. However, CD45 has recently been shown to dephosphorylate the positive regulatory tyrosine of the Lck and Lyn PTKs, thus inhibiting their activity (29, 30). Hence, in thymocytes from CD45-null mice, Lck is hyperphosphorylated not only on the negative regulatory tyrosine residue (26), but also on the positive regulatory tyrosine, resulting in a net increase in kinase activity, as determined by in vitro kinase assays (30). These data are difficult to reconcile with recent reports that T cell development in CD45-null mice can be rescued by the introduction of a constitutively active mutant of Lck (LckY505F; Refs. 31 and 32). The most likely explanation for these contradictory results is that, in the absence of CD45, Lck is maintained in a closed conformation due to interaction of the SH2 domain with the positive regulatory phosphotyrosine. This in turn might prevent Lck from interacting with its substrates in vivo. In contrast, in vitro kinase experiments, which rely on the use of short peptide substrates to assess kinase activity, might not reflect the inability of full-sized substrates to interact with Lck. Therefore, the net effect of CD45 in vivo might be to activate, rather than inhibit, the activity of Src-family PTKs. The results presented in this paper demonstrate that a substantial fraction of thymocytes expressing autoreactive TCRs escape negative selection in CD45-null mice, which must result from diminished signaling through the TCR. Hence, our results support a positive role for CD45 in regulating TCR signaling.

As discussed above, previous work demonstrated that CD45 plays a crucial role in both positive and negative selection in the thymus. However, due to the use of TCR transgenes or to ex vivo experimental approaches, none of these studies could directly test the hypothesis that a CD45 deficiency can lead to the production of mature T cells expressing TCRs with high affinity for self-MHC-peptide complexes. In contrast, our approach of analyzing the specificity of the TCRs expressed by peripheral T cells from CD45-null mice yields an accurate assessment of the ability of CD45 to affect the outcome of thymic selection. Indeed, thymic selection in CD45-null mice is mediated by naturally occurring MHC-peptide ligands, and operates in an intact thymic microenvironment on thymocytes expressing normal levels of TCR (19, 20). Moreover, the frequency (55%) of T cells from CD45-null mice that were found to react against self-MHC-peptide ligands is probably lower than the actual frequency of autoreactive cells that would normally have been deleted during thymic selection, because negative selection of thymocytes requires a lower threshold of activation than mature T cell responses (67).

The high frequency (7/11 or 64%) of alloreactive hybridomas observed in our study is far greater than the already relatively high frequency with which TCRs react with foreign MHC molecules. Indeed, an estimated 1–10% of mature T cells are usually expected to react with an allogeneic MHC molecule presenting one or more of the ~2000 peptides that may be bound to that molecule on the surface of a cell (68, 69, 70). Such high incidence of alloreactivity suggests that, in the absence of CD45, only TCRs capable of strong interaction with MHC amino acids can generate sufficiently strong signals to trigger positive selection. This implies that increased affinity for MHC can compensate for debilitated TCR signaling. Moreover, because MHC alleles tend to differ in amino acids that contact peptide rather than those that contact TCR (71, 72, 73, 74), it follows that TCRs selected in the context of a CD45 deficiency should be biased toward recognition of allogeneic MHC, especially those MHC alleles most closely related to the positively selecting MHC. The same phenomenon has been observed previously in two other experimental models where a decrease in the density or in the diversity of MHC-peptide ligands presented within the thymus prevented the negative selection of T cells that could react against APCs presenting normal levels of self-MHC, or a diverse repertoire of self-MHC-peptide ligands, respectively (11, 75). Thus, our results are consistent with the peptide-oriented model of selection proposed by Ignatowicz et al. (11), whereby TCRs that react primarily with peptide amino acids mediate successful positive selection, and TCRs that react primarily with MHC amino acids trigger negative selection.

It must be noted that the T cells bearing self-reactive TCRs that develop as a consequence of impaired TCR-mediated signaling cannot trigger autoimmune responses in the organism from which they originate, because the signaling deficiency that initially prevented their negative selection persists after they have emigrated to the periphery. However, tolerance to self may be broken if the signaling deficiency is compensated by supplying a functional copy of the defective gene, as we and others (17) have shown, but could conceivably also be compensated by any intervention aimed at increasing the strength of TCR signals. Hence, autoreactivity may result whether a TCR signaling defect occurs only transiently during thymic development, or whether the defect is mended after the avidity of the TCR for self has been calibrated by intra or extrathymic selection processes. Therefore, caution should be exercised in the device of therapies for immune disorders pertaining to T cell function, because improving T cell recognition/activation events under these circumstances might lead to autoimmunity. Accordingly, therapies for T cell immunodeficiency must be targeted at earlier stages of T cell development, ideally at the self-renewing hemopoietic progenitors.

Our results demonstrate for the first time that negative selection is impaired in the absence of CD45, such that mature T cells are produced that express TCRs with high reactivity toward self-MHC-peptide ligands. These findings show conclusively that CD45 is directly involved in the shaping of the T cell repertoire. Moreover, our observation that the threshold of selection is increased in CD45-null mice clearly establishes CD45 as a positive regulator of TCR signaling.


   Acknowledgments
 
We thank S. Lesage for technical assistance, and Dr. J. C. Zúñiga-Pflücker (Department of Immunology, University of Toronto, Toronto, Ontario, Canada) for critical review of this manuscript.


   Footnotes
 
1 This work was supported by the Medical Research Council of Canada and by PROCREA BioSciences. S.T. is the recipient of a Doctoral Research Award from the Medical Research Council of Canada. Back

2 Address correspondence to Dr. P. Hugo, PROCREA BioSciences, 6100 Royalmount, Montréal, Québec, Canada, H4P 2R2. Back

3 Abbreviations used in this paper: DP, double-positive (CD4+CD8+); SP, single-positive (CD4+CD8- or CD4-CD8+); PTK, protein tyrosine kinase. Back

Received for publication October 12, 1999. Accepted for publication July 3, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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