PEST Domain-Enriched Tyrosine Phosphatase (PEP) Regulation of Effector/Memory T Cells

Protein tyrosine phosphatases (PTPases) play important roles in activation and termination of T cell–antigen receptor (TCR) signaling, as well as virulence factors encoded by infectious organisms that enable evasion of protective host responses (1). PEST domain–enriched tyrosine phosphatase (PEP) is a cytoplasmic PTPase expressed exclusively in hematopoietic cells (2) and associates with Csk (C-terminal Src tyrosine kinase) to inhibit TCR signaling through dephosphorylation of the autophosphorylation site (Tyr³⁹⁴) of the Lck protein tyrosine kinase (3–6). To understand the role of PEP in T cell development and differentiation, pep^–/– mice were generated in which PEP protein expression was specifically ablated (fig. S1, A and B) (7).

Thymocyte number and subsets were similar in pep^–/– and wild-type mice (Fig. 1A, table S1); the only discernible differences were a marginal increase in CD5 expression in pep^–/– CD4⁺CD8⁺ [double-positive (DP)] thymocytes and a small increase (∼30%) in CD4⁺ pep^–/– T cell number. Because increased CD5 expression at the DP stage is associated with enhanced TCR signaling (8, 9), we examined the positive and negative selection of pep^–/– thymocytes. pep^–/– female mice expressing a major histocompatability complex (MHC) class I–restricted TCR transgene specific for the male H-Y antigen (10), developed increased numbers of H-Y TCR⁺ CD8⁺ T cells in the thymus and peripheral lymphoid compartments (Fig. 1B, fig. S1C). Similarly, pep^–/– mice expressing an MHC class II–restricted DO11.10 TCR transgene specific for ovalbumin (OVA) displayed an increase in the development of CD4⁺ T cells (Fig. 1C, fig. S1D) (11). However, the absence of PEP had no detectable effect on negative selection, as measured by normal deletion of H-Y TCR⁺ CD8⁺ thymocytes in male pep^–/– mice (12) or polyclonal deletion of DP thymocytes by treating with a monoclonal antibody (mAb) against CD3ϵ (fig. S1, E and F) (13). Together, these data support a differential inhibitory role of PEP on positive and negative selection.

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Although young (4- to 6-week-old) transgenic pep^–/– mice lacking a TCR transgene demonstrated relatively small alterations in peripheral lymphoid organs, older (>6 months) pep^–/– mice developed splenomegaly and lymphadenopathy (Fig. 1D; fig. S2, A to C; and table S1). In particular, older pep^–/– mice had increased numbers of T cells within the effector/memory T cell pool as determined by CD4⁺CD44^hiCD62L^lo and CD45RB^hi expression (Fig. 2A) (14, 15). A similar accumulation of effector/memory pep^–/– T cells was also evident within the CD8 compartment of older animals, with increased numbers of CD44^hiCD62L^lo-hi cells (Fig. 2B). An absolute increase in both effector (CD43-1B11^hi) and, to a lesser extent, memory (CD43-1B11^lo) CD8⁺ cell number was observed (fig. S2D) (16).

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We next examined the mechanisms by which PEP deficiency leads to the observed accumulation of effector/memory T cells. After the first 2 days of TCR activation, wild-type and pep^–/– naïve (CD44^loCD62L^hi) T cells demonstrated comparable growth and cycling (Fig. 3, A and B). In addition, these cells demonstrated normal TCR-mediated [³H]thymidine incorporation, cytokine production, mobilization of intracellular calcium concentration ([Ca²⁺]_i), expression of activation markers (e.g., CD25), and tyrosine phosphorylation of cellular proteins (Fig. 3, C to E; fig. S3, C and D) (14). These results indicate that the absence of PEP had minimal functional or apparent biochemical consequences on the initial stages of activation of naïve T cells.

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In contrast to the first 2 days of naïve T cell activation, pep^–/– CD4⁺ and CD8⁺ T cells demonstrated a 2.5-fold growth advantage, which was also reflected by increased cell cycling, during days 3 and 4 after T cell activation (Fig. 3, A and B). Enhanced cycling on day 4 was observed in cells isolated from both young and old pep^–/– mice (fig. S3A) and was not due to differences in rates of activation-induced cell death (fig. S3B). These growth differences and the accumulation of effector/memory T cells in pep^–/– mice prompted us to further analyze the consequences of PEP deficiency on T cell differentiation.

Naïve T cells, activated in vitro using mAbs against CD3ϵ and CD28 for 3 days, were rested for two additional days to generate an effector cell population (17). Upon TCR restimulation, these effector cells derived from pep^–/– naïve T cells displayed increased proliferation and cytokine production (Fig. 3F and fig. S3C). These differences not only reflected greater expansion of pep^–/– cells, but also enhanced cellular signaling as measured by higher mean fluorescence intensity (MFI) of CD25 and increased ability to flux [Ca²⁺]_i (Fig. 3, G to H). Because PEP regulates Lck autophosphorylation (3, 18), we analyzed the phosphorylation status of the autoregulatory (Tyr³⁹⁴) and inhibitory (Tyr⁵⁰⁵) tyrosines within Lck (19). Although the Lck Tyr³⁹⁴ site in wild-type effector T cells demonstrated transient TCR-inducible phosphorylation, this was enhanced and sustained in pep^–/– effector cells (Fig. 3I). In contrast, no consistent change in Tyr⁵⁰⁵ phosphorylation was detected. As a consequence, phosphorylation of ZAP-70 at Tyr³¹⁹ and mitogen-activated protein kinase (MAPK) activation were prolonged and enhanced (Fig. 3I and fig. S3E). The normal cycling of pep^–/– effector/memory cells in response to other stimuli (interleukin IL-2 or IL-15 receptors), which are also regulated by Lck (20), indicates that the inhibitory role of PEP appears confined to the TCR signaling pathway (fig. S3F). Together, these data support the notion that PEP controls both proliferation and functions of activated effector T cells through dephosphorylation of the Lck autoregulatory catalytic site.

We next examined whether freshly isolated effector/memory cells demonstrated similarly enhanced TCR functions as in vitro generated effector cells. CD44^hiCD62L^lo pep^–/– effector/memory T cells, isolated from 3-month-old mice, demonstrated similarly enhanced TCR-mediated proliferation and cell cycle divisions as compared with wild-type cells (Fig. 4A). To further dissect the in vivo mechanisms by which PEP deficiency generates increased effector/memory cells, we used the DO11.10 TCR⁺ system to transfer wild-type or pep^–/– naïve DO11.10 TCR ⁺ T cells to irradiated BALB/c mice. Three days after OVA peptide immunization, there were three times as many pep^–/– T cells in the draining lymph node as wild-type T cells (Fig. 4B). A sixfold expansion of pep^–/– T cells was observed on day 5 and correlated with increased cell division of pep^–/– splenic T cells (Fig. 4, B and C). As with in vitro generated effector T cells (Fig. 3G), in vivo generated effector cells revealed increased numbers of CD25⁺ cells and higher MFI of CD25 upon in vitro rechallenge with antigen (Fig. 4D). Thus, enhanced proliferation in the absence of PEP occurs in vivo, in an antigen-dependent fashion.

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Consistent with unaffected activation-induced cell death observed in pep^–/– T cells in vitro (fig. S3B), the rate of contraction after OVA challenge was similar between pep^–/– and wild-type T cells in vivo. By day 15 after antigen challenge, the ratio of remaining numbers of pep^–/– relative to wild-type T cells was comparable to that observed on day 5 (Fig. 4B). The differential effects on effector and naïve T cells by PEP deficiency may reflect the coregulated expression of PEP and the closely related PTP-PEST during T cell differentiation (14, 21). Additional studies of mice deficient in both PTPases will help to test this hypothesis.

The enhanced immune functions observed in pep^–/– T cells were also associated with spontaneous development of germinal centers (GCs) in the spleens and Peyer's patches of these animals (Fig. 4E). Wild-type mice generally demonstrate small numbers of immature GCs (22). In contrast, pep^–/– animals demonstrated increased numbers of large, well-formed GCs, with a corresponding increase in the numbers of GL-7⁺ B cells, immunoglobulin IgG1⁺ foci, and intermediate CD21 (CD21^int) CD23⁺ follicular B cells (14). Analysis of signaling mediated by the B cell–antigen receptor (BCR), however, revealed minimal intrinsic biochemical and functional alterations in splenic B cells derived from pep^–/– mice (fig. S4, A to C). Complementation studies with adoptive transfer experiments using pep^–/– and wild-type T and B cells may reveal additional functions of PEP within the B cell compartment. Nonetheless, GC formation in pep^–/– mice depended on cooperation between T and B cells, as administration of a blocking CD40L-specific mAb disrupted GC formation (14), demonstrating that the B cell effects were, in part, secondary to enhanced pep^–/– T cell functions.

Germinal centers serve as important sites for immune dysregulation in the pathogenesis of autoimmune disorders (22), and spontaneous GC formation has been observed in a number of mouse strains that develop autoimmune disorders or have altered B cell function (23–26). Although pep^–/– mice demonstrated increased serum levels of IgG1, IgG2a, and IgE, neither young nor old pep^–/– mice demonstrated any increased incidence of autoantibodies or evidence of autoimmune-mediated organ damage, as compared with wild-type littermates (Fig. 4, F and G) (14). This uncoupling of spontaneous GC formation from autoimmune stigmata indicates a stepwise requirement for immune cell dysregulation for the development of clinical manifestations of autoimmune disorders. Because PEP deficiency does not appear to affect the deletion of autoreactive T cells, additional defects in central or peripheral tolerance are likely required as initiators of some autoimmune disorders. In the context of these initiating immune defects, enhanced expansion of autoreactive effector/memory T cells, as manifested by PEP deficiency, could culminate in clinical autoimmunity.