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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2008 Feb 19;105(8):2987–2992. doi: 10.1073/pnas.0708381104

Tpl2 and ERK transduce antiproliferative T cell receptor signals and inhibit transformation of chronically stimulated T cells

Christos Tsatsanis *,†, Katerina Vaporidi ‡, Vassiliki Zacharioudaki *,§, Ariadne Androulidaki *, Yuri Sykulev , Andrew N Margioris *, Philip N Tsichlis ‡,†
PMCID: PMC2268572  PMID: 18287049

Abstract

The protein kinase encoded by the Tpl2 protooncogene plays an obligatory role in the transduction of Toll-like receptor and death receptor signals in macrophages, B cells, mouse embryo fibroblasts, and epithelial cells in culture and promotes inflammatory responses in animals. To address its role in T cell activation, we crossed the T cell receptor (TCR) transgene 2C, which recognizes class I MHC presented peptides, into the Tpl2−/− genetic background. Surprisingly, the TCR2Ctg/tg/Tpl2−/− mice developed T cell lymphomas with a latency of 4–6 months. The tumor cells were consistently TCR2C+CD8+CD4−, suggesting that they were derived either from chronically stimulated mature T cells or from immature single positive (ISP) cells. Further studies showed that the population of CD8+ ISP cells was not expanded in the thymus of TCR2Ctg/tg/Tpl2−/− mice, making the latter hypothesis unlikely. Mature peripheral T cells of Tpl2−/− mice were defective in ERK activation and exhibited enhanced proliferation after TCR stimulation. The same cells were defective in the induction of CTLA4, a negative regulator of the T cell response, which is induced by TCR signals via ERK. These findings suggest that Tpl2 functions normally in a feedback loop that switches off the T cell response to TCR stimulation. As a result, Tpl2, a potent oncogene, functions as a tumor suppressor gene in chronically stimulated T cells.

Keywords: CTLA4, cancer, lymphoma, T cell receptor transgene 2C (TCR2C), CD8

Antigens are proteolytically cleaved by antigen-presenting cells such as dendritic cells, macrophages, and B cells, and after cleavage, they are presented in complex with MHC surface molecules (1). Antigenic peptides associated with and presented by class I MHC molecules are recognized by CD8 single-positive (SP) T cells, whereas antigenic peptides presented by class II MHC molecules are recognized by CD4+ T cells (1). T cell receptor (TCR) stimulation by antigen-MHC complexes, in combination with costimulatory molecules expressed by antigen-presenting cells, promotes the activation of T cells and the initiation of the immune response (1).

Activated T cells deliver cytotoxic signals or provide help for the activation of B cells (2), and once their mission is accomplished, their activity is curtailed. The turning off of the T cell response is critical for organismal homeostasis and depends on the induction of several molecules that are involved in the initiation of inhibitory feedback loops. Prominent among them are membrane proteins such as CTLA4 and PD-1 (3), as well as several secreted, cytoplasmic, or nuclear signaling proteins, including cytokines such as TGF-β and TNF-α (4, 5), transcription factors such as Foxj1, Foxp3, and LKLF (6–8), E3 ubiquitin ligases such as Cbl-b and GRAIL (9, 10), and kinases such as DRAK2 (11).

CTLA4, a member of the CD28 coreceptor family, which includes CD28, the inducible costimulator (ICOS), CTLA4, PD-1, and BTLA (12, 13), is expressed at very low levels in unstimulated T cells, and it is induced within 24 h from the start of the stimulation. CTLA4 is recognized by the ligands B7–1 (CD80) and B7–2 (CD86), which also recognize CD28. However, B7–1 and B7–2 exhibit significantly higher affinities for CTLA4 than for CD28. Because CTLA4 stimulation gives rise to inhibitory T cell signals, the induction of CTLA4 inhibits T cell activation, both indirectly, by outcompeting CD28, and directly (12, 13).

Direct inhibition depends in part on the cross-talk between CTLA4 and CD28, which is mediated by PP2A. The protein phosphatase PP2A binds constitutively the intracellular domain of CTLA4, and it is released after TCR-CTLA4 coligation (14). The released PP2A may bind CD28 to inhibit PI-3K and Akt activation (13, 15). In addition, it dephosphorylates Akt and inhibits the activation of Rap1 and Cbl-b by CD28 signals (13). Other direct inhibitory mechanisms include the disruption of membrane lipid rafts (16) and potentially the induction of TGF-β (17). The preceding mechanisms of inhibition of T cell activation by CTLA4 are cell autonomous. However, CTLA4 may also inhibit the activation of effector T cells by non-cell-autonomous mechanisms (12). Thus, CTLA4 in Tregs and soluble forms of CTLA4 bind CD80 and CD86 on the surface of effector T cells or dendritic cells and inhibit their activation by reverse signaling (18, 19).

The Tpl2 protooncogene encodes a serine–threonine protein kinase that is activated by provirus integration in retrovirus-induced T cell lymphomas and mammary adenocarcinomas in rodents. Overexpression of Tpl2 in a variety of cell types activates ERK, JNK, p38MAPK, NFAT, and NF-κB; promotes cell proliferation; and induces cell transformation (20–23). Moreover, overexpression of Tpl2 induces IL-2 expression in T cell lines (24, 25) and tumors in mice (23).

In this article, we demonstrate that contrary to expectations, knocking out Tpl2 enhances the cellular response to T cell receptor signals by partially blocking a CTLA4-dependent inhibitory feedback loop. Chronic stimulation of CD8 SP T cells in Tpl2−/− mice expressing the TCR2C transgene gives rise to CD8 SP T cell lymphomas.

Results

Tpl2−/− Mice Harboring the TCR2C Transgene Develop CD8+ T Cell Lymphomas.

To determine the role of Tpl2 in T cell signaling, we crossed the TCR transgene 2C (26) into the Tpl2−/− genetic background. TCR2Ctg/tg/Tpl2+/+ and TCR2Ctg/tg/Tpl2−/− mice developed normally, and in both, the great majority of CD8 SP T cells expressed the TCR2C transgene [Fig. 1E and supporting information (SI) Fig. 6B]. Moreover the ablation of Tpl2 did not affect the splenic ratio of CD4/CD8 SP T cells in young, 4- to 6-week-old mice (SI Fig. 6A). To our surprise, however, the majority of TCR2Ctg/tg/Tpl2−/− mice developed T cell lymphomas, characterized by both thymic and splenic enlargement, at 4–6 months of age. Interestingly, the TCR2Ctg/tg/Tpl2+/+ mice, as well as the nontransgenic Tpl2+/+ and Tpl2−/− control mice, did not develop tumors, whereas the TCR2Ctg/tg/Tpl2+/− mice developed tumors with a longer latency than that of the TCR2Ctg/tg/Tpl2−/− mice (Fig. 1A), suggesting that tumor induction by the TCR transgene depends on the Tpl2 dosage.

Fig. 1.

Fig. 1.

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TCR2Ctg/tg/Tpl2−/− mice develop CD8+ T cell tumors. (A) Tumor incidence at the end of the monitoring period (10 months) (Left) and timing of tumor detection (Kaplan Meyer survival curves) (Right) revealed significant differences between TCR2Ctg/tg/Tpl2+/+ and both TCR2Ctg/tg/Tpl2+/− and TCR2Ctg/tg/Tpl2−/− mice as determined by log rank analysis (P < 0.0001). (B) H&E staining and immunohistochemical analysis of a thymoma developing in a TCR2Ctg/tg/Tpl2−/− mouse. Data are representative of eight thymomas similarly analyzed. Ig isotype (Ig) control is shown in Lower Right. (C) H&E and immunohistochemical staining of an enlarged spleen from a TCR2Ctg/tg/Tpl2−/− mouse. Splenic architecture remained intact. CD3+ T cells were localized primarily in the white pulp. However, CD3+ T cells were also detected in a diffuse pattern within the red pulp. These data were representative of eight spleens similarly analyzed. (D) CD4/CD8 staining and flow cytometry of thymocytes (Left) and splenocytes (Right) from a tumor-bearing TCR2Ctg/tg/Tpl2−/− mouse. Similar data were obtained from eight mice similarly analyzed. (E) The great majority of CD8 SP thymocytes express the TCR2C transgene. Thymocytes were stained with FITC-conjugated anti-CD8 and with anti-TCR2C combined with an FITC-labeled anti-mouse secondary antibody. (F) Tpl2 ablation does not induce a developmental arrest at the ISP stage in developing thymocytes in TCR2Ctg/tg/Tpl2−/− mice. Cells were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD24 (HSA), and anti-CD5. CD3modCD8+CD4− cells were gated and analyzed for the expression of CD5 and CD24 (HSA). Data are representative from a total of four animals per strain analyzed.

Histologic and immunohistochemical analysis of the thymic tumors revealed a diffuse pattern of lymphoid cells that stained positive for both CD3 and the TCR transgene 2C (Fig. 1B). The enlarged spleens of the same mice, however, retained the normal splenic architecture. CD3+ T cells were localized primarily in the white pulp, as expected. However, CD3+ T cells were also detected in a diffuse pattern within the red pulp (Fig. 1C). Staining of single-cell suspensions of tumor cells from eight tumors with anti-CD4 and anti-CD8 revealed that all of the tumors are composed of CD8 SP T cells (Fig. 1D). The CD8 SP phenotype suggests that the cells of origin of the tumors may be the mature CD8 SP T cells (27) or the immature single positive thymocytes (ISPs). If the tumors arise from the ISPs, one would expect accumulation of ISPs in the thymus of preleukemic TCR2Ctg/tg/Tpl2−/− mice (28). Staining of CD8 SP thymocytes from four TCR2Ctg/tg/Tpl2+/+ and four TCR2Ctg/tg/Tpl2−/− mice for CD3, HSA, and CD5, which are differentially expressed in mature CD8 SP thymocytes and ISPs (28), revealed that the number of thymocytes that exhibit the ISP phenotype (CD3modCD8+CD4−HSA+CD5−) was similar in both strains. This finding suggests that the TCR2Ctg/tg/Tpl2−/− tumors may develop via the continuous stimulation of mature CD8 SP T cells.

The TCR2C transgene is expressed early in T cell development (26) and continues to be expressed in mature T cells. SI Fig. 6B shows that the great majority of CD8 SP splenocytes express the TCR2C transgene. Moreover, tumor cells exhibit a CD8 SP phenotype, and they express TCR2C, suggesting that expression of the transgene contributes to tumorigenesis. This finding provides additional support to the conclusion that tumors develop via the continuous TCR stimulation of CD8 SP T cells. The stimulus could be provided by endogenous peptides, such as dEV8, which are presented by class I MHC and are known to trigger TCR2C (29).

T Cells of Tpl2−/− and TCR2Ctg/tg/Tpl2−/− Mice Exhibit Enhanced Proliferation Upon TCR Stimulation.

Given that the tumors arise only in Tpl2−/− mice, we hypothesized that Tpl2 ablation may enhance the proliferative capacity of the responding CD8 SP T cells. To test this hypothesis, splenocyte preparations isolated from TCR2Ctg/tg/Tpl2−/− and TCR2Ctg/tg/Tpl2+/+ mice were stimulated with anti-CD3, anti-CD3 plus anti-CD28, the peptide SIY, or the low-affinity self-peptide dEV8. [3H]Thymidine incorporation, measured 48 and 72 h from the start of the stimulation, revealed enhancement of cell proliferation in Tpl2−/− cells (Fig. 2A). Increased proliferation was also observed in anti-CD3-/anti-CD28-stimulated splenocytes from Tpl2−/− mice not harboring the TCR2C transgene, suggesting that the enhanced proliferation is transgene-independent (SI Fig. 7).

Fig. 2.

Fig. 2.

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Tpl2 ablation promotes T cell proliferation. (A) [3H]Thymidine incorporation was measured in splenocytes stimulated with anti-CD3, anti-CD3 and anti-CD28, SIY (10−6 M), or dEV8 (10−6 M). Data represent the average of four mice per strain. *, P < 0.05; **, P < 0.001. (B) Proliferation of CD8 SP splenic T cells loaded with CFSE and stimulated with SIY (10−6 M). Cells were stained with anti-CD8 PE and visualized by flow cytometry 2–5 days after stimulation. The results shown are representative of four mice analyzed per strain. (C) CD8 SP T cells were isolated and stimulated by SIY-loaded irradiated feeder splenocytes from C57BL/6 mice. Proliferation was measured by [3H]thymidine incorporation. Data represent the average of four mice per strain. (D) TCR2Ctg/tg/Tpl2−/− splenocytes exhibit a lower activation threshold than TCR2Ctg/tg/Tpl2+/+ splenocytes. Splenocytes (2 × 105) were stimulated with increasing concentrations of SIY. CD69 expression was measured by flow cytometry 12 h later. Data are representative of four mice per strain analyzed.

In parallel experiments, splenocytes were labeled with 5-(and 6-)carboxyfluoroscein diacetate succimidyl ester (CFSE), and 18 h later, they were stimulated with the peptide SIY. CFSE fluorescence intensity, measured in CD8 SP T cells by flow cytometry, revealed that TCR2Ctg/tg/Tpl2−/− cells respond to proliferative TCR signals more rapidly, and they undergo more cell divisions over a 5-day period than cells from TCR2Ctg/tg/Tpl2+/+ mice (Fig. 2B).

It is theoretically possible that the higher numbers of cells undergoing multiple cell divisions in SIY-stimulated TCR2Ctg/tg/Tpl2−/− cell cultures (see Fig. 2B) may be due to a reduction in the rate of apoptosis. To test this hypothesis, we used Annexin V to stain anti-CD3-/anti-CD28-stimulated Tpl2+/+ and Tpl2−/− CD8 SP T cells. The results revealed no differences in the rate of apoptosis between these cell populations (SI Fig. 8), suggesting that the higher number of dividing cells in the Tpl2−/− population is due to enhanced proliferation rather than to reduced apoptosis.

To determine whether CD8 cell hyperproliferation is an intrinsic property of the Tpl2 knockout cells, we carried out [3H]thymidine incorporation assays on purified CD8 SP T cells from TCR2Ctg/tg/Tpl2−/− and TCR2Ctg/tg/Tpl2+/+ mice, stimulated with the SIY peptide. Stimulation carried out by using C57BL/6-derived feeders loaded with the peptide showed that TCR2Ctg/tg/Tpl2−/− CD8 SP T cells indeed exhibit enhanced proliferation (Fig. 2C), suggesting that hyperproliferation is due to cell-autonomous mechanisms.

The preceding data also suggested that Tpl2−/− CD8 SP T cells may exhibit a decreased activation threshold. This hypothesis was confirmed by staining splenocytes from TCR2Ctg/tg/Tpl2+/+ and TCR2Ctg/tg/Tpl2−/− mice, stimulated with increasing concentrations of the SIY peptide for the activation marker CD69, which revealed that CD69 is induced by lower peptide concentrations in TCR2Ctg/tg/Tpl2−/− cells (Fig. 2D).

Tpl2 Transduces T Cell Activation Signals That Regulate the Expression of Inhibitory Molecules.

T cells responding to T cell activation signals express a variety of molecules that activate negative feedback loops that limit the T cell response (see Introduction). One of these molecules, CTLA4, interacts with high affinity with the molecules CD80 (B7–1) and CD86 (B7–2) and delivers a strong antiproliferative signal (3). To determine the mechanism by which Tpl2 inhibits T cell proliferation, we examined the expression of CTLA4, IL-2Rα, TGF-β, and TNF-α in Tpl2+/+ and Tpl2−/− T cells before (Fig. 3A) and 48 h after exposure to T cell activation signals. In Fig. 3B, the cells were stimulated with ConA plus IL-2; in Fig. 3C, with anti-CD3 and anti-CD28; and in Fig. 3D, with the peptide SIY. In a separate experiment, cells were stimulated with the self-peptide dEV8 (Fig. 3E). In all cases, the ablation of Tpl2 inhibited the induction of CTLA4, suggesting that CTLA4 induction depends on activation signals transduced via Tpl2.

Fig. 3.

Fig. 3.

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T cells from TCR2Ctg/tg/Tpl2−/− mice express lower levels of CTLA4 upon activation. Total splenocytes were unstimulated (A), stimulated with ConA plus IL-2 (B), stimulated with anti-CD3 plus anti-CD28 (C), or stimulated with the peptide SIY (D), and expression of CTLA4 was measured 48 h after stimulation. (E) Total splenocytes were stimulated with dEV8, stained for CTLA4 72 h later, and analyzed by flow cytometry.

TCR2Ctg/tg/Tpl2+/+ and TCR2Ctg/tg/Tpl2−/− T cells stimulated with the peptide SIY or with anti-CD3 plus anti-CD28 express similar levels of TNF-α and TGF-β, (SI Fig. 9 A and B), as well as IL-2Rα and IL-2 (SI Fig. 9C and data not shown). These data collectively suggest that the T cell activation signals transduced by Tpl2 target CTLA4, and that inhibition of CTLA4 induction in TCR-stimulated Tpl2−/− T cells may be largely responsible for the enhanced proliferation of these cells in response to TCR signals.

Tpl2 Is Required for the Transduction of TCR Signals That Activate MEK and ERK but Not p38MAPK or NF-κB in T Cells.

To determine the signaling defects that are responsible for the inhibition of CTLA4 induction in TCR-stimulated Tpl2−/− T cells, we stimulated CD3+ T cells from TCR2Ctg/tg/Tpl2+/+ and TCR2Ctg/tg/Tpl2−/− mice via the TCR, and we examined the activation of ERK1/ERK2, MEK1 and MEK2, and NF-κB. The cells were isolated by negative selection from spleens and they were treated with the peptide SIY immediately after plating (Fig. 4 A1 and B1). In parallel experiments, total splenocytes were stimulated with ConA and cultured in IL-2-containing media for 3 days. The cells were then starved of IL-2 for 4 h, and they were stimulated with anti-CD3 or with SIY (Fig. 4 A2 and B2). ERK1/2 (Fig. 4 A1 and A2), MEK1/2 (Fig. 4 B1 and B2), and p38MAPK (SI Fig. 10A) activation was monitored by probing Western blots of lysates, harvested before and at sequential time points after stimulation, with phospho-specific antibodies. NF-κB activation was monitored by EMSAs (SI Fig. 10B). The results revealed that, whereas Tpl2 is required for full activation of MEK and ERK, it is dispensable for the activation of p38MAPK and NF-κB by TCR signals.

Fig. 4.

Fig. 4.

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Tpl2 ablation partially blocks the phosphorylation of ERK1, ERK2, MEK1, and MEK2 in response to TCR signals. CD3+ T cells isolated from spleens were stimulated with anti-CD3 or SIY (A1 and B1) or spleen cells were plated in the presence of ConA and IL-2 for 3 days, starved from ConA and IL-2 for 4 h and stimulated with anti-CD3 or SIY (A2 and B2). Western blots were probed with phosphospecific and total ERK1/2 or MEK1/2 antibodies. Histograms represent the average of four independent experiments [P < 0.01 (**) and P < 0.001 (***) compared with the wild-type sample exposed to the same stimulus for the same time period].

The Induction of CTLA4 by TCR Signals Is ERK-Dependent.

To determine whether the activation of ERK by Tpl2-transduced signals is required for the induction of CTLA4, TCR2Ctg/tg/Tpl2+/+ and TCR2Ctg/tg/Tpl2−/− cells were stimulated with anti-CD3 and anti-CD28 or SIY. Half of the cultures were treated with the MEK inhibitor UO162 before stimulation. Forty-eight hours later, cells were stained with the anti-CTLA4 antibody, and they were analyzed by flow cytometry. The results showed that the MEK inhibitor substantially inhibited the induction of CTLA4 in Tpl2+/+ cells, but it had no effect on the Tpl2−/− cells. We conclude that ERK activation is required for the induction of CTLA4 (Fig. 5 A and B).

Fig. 5.

Fig. 5.

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Expression of CTLA4 depends on activation of MEK. Spleen cells were stimulated for 48 h with anti-CD3 and anti-CD28 (A) or SIY (B) in the presence or absence of the MEK inhibitor UO126. CTLA4 expression in CD8 SP T cells was measured by flow cytometry. Results are representative of three independent experiments with three mice per strain. (C) Inhibition of MEK results in increased proliferation of CD8 SP T cells. Total splenocytes were loaded with CFSE and stimulated for 2, 3, or 5 days with SIY (10−6 M). Proliferation of CD8 SP T cells was measured by flow cytometry. Results are representative of two independent experiments with three mice per strain. (D) Total splenocytes were isolated from four 6- to 8-week-old mice (two mice of each genotype) and four 6-month-old mice (two mice of each genotype), and CTLA4 expression was measured. The two 6-month-old TCR2Ctg/tg/Tpl2−/− mice had developed tumors at the time of experiment. Shown are representative data of a total of four animals per strain from the analysis of the splenocytes of 6- to 8-week-old animals and of one tumor derived from a 6-month-old TCR2Ctg/tg/Tpl2−/− mouse.

ERK Activation Inhibits T Cell Proliferation in Response to TCR Signals.

To confirm that Tpl2-transduced ERK activation signals limit the proliferation of T cells in response to TCR stimulation, CFSE-labeled total splenocytes from TCR2Ctg/tg/Tpl2+/+ mice were stimulated with the SIY peptide before and after pretreatment for 1 h with UO162. Flow-cytometric analysis of the CD8 SP T cells revealed that inhibition of the MEK/ERK pathway is associated with a significant increase in the number of divisions cells undergo after TCR stimulation. We conclude that, after its activation by Tpl2-transduced TCR signals, ERK inhibits T cell proliferation by promoting the induction of CTLA4. As a result, inhibition of the MEK/ERK pathway enhances the proliferative capacity of the cells (Fig. 5C).

Tpl2 Regulates the Expression of CTLA4 in Vivo.

To determine the biological significance of CTLA4 regulation by Tpl2 in vivo, we addressed whether CD8 SP splenic T cells from TCR2Ctg/tg/Tpl2+/+ and TCR2Ctg/tg/Tpl2−/− mice differ in the expression of CTLA4 in vivo. To this end, splenocytes from four 6- to 8-week-old (two TCR2Ctg/tg/Tpl2+/+ and two TCR2Ctg/tg/Tpl2−/−) and four 6-month-old (two TCR2Ctg/tg/Tpl2+/+ and two TCR2Ctg/tg/Tpl2−/−) animals were stained with anti-CD8 and anti-CTLA4. Both 6-month-old TCR2Ctg/tg/Tpl2−/− animals had developed T cell lymphomas at the time of the experiment. The expression of CTLA4 was examined in CD8 SP T cells by flow cytometry. The representative data shown in Fig. 5D demonstrate that splenocytes from both the TCR2Ctg/tg/Tpl2+/+ and the TCR2Ctg/tg/Tpl2−/− mice express CTLA4 at 6–8 weeks, although the level of expression was lower in the Tpl2−/− mice. Tumors developing in the same mice were negative for CTLA4 expression. We conclude that Tpl2 is required for full induction of CTLA4 not only in culture but also in animals and that tumor selection favors cells that express the lowest possible levels of CTLA4.

Discussion

The activation of T cells by antigen-induced TCR signals is a self-limiting process. After the initial response, a number of molecular mechanisms are set in motion to limit T cell activation. Several signaling molecules that contribute to this process by activating feedback inhibitory loops have been identified to date (see Introduction). Failure of these inhibitory mechanisms may have detrimental effects to the homeostatic balance of the organism because it may give rise to autoimmunity or cancer (3).

The hyperesponsiveness of the CD8 SP T cells of TCR2Ctg/tg/Tpl2−/− mice to TCR signals indicates that the physiological role of Tpl2, a kinase that promotes T cell proliferation and oncogenesis when overexpressed in T cells (23, 30, 31), is to transmit negative regulatory signals that suppress T cell proliferation. The inhibitory signals are generated by CTLA4, and perhaps other, as-yet-unidentified inhibitory molecules, which are induced during T cell activation by Tpl2.

In this article, we presented evidence that Tpl2 is required for full activation of ERK, but not p38MAPK and NF-κB, by TCR signals, and that the activation of ERK in response to these signals is a prerequisite for the induction of CTLA4. As a result, the induction of CTLA4 by TCR signals in mature CD8 SP T cells of TCR2Ctg/tg/Tpl2−/− mice is weak. The same mice develop CD8 SP T cell lymphomas. The surface phenotype of the tumor cells suggests that they may derive from mature CD8 SP T cells, or from ISPs. Derivation of the tumor cells from ISPs would be likely if Tpl2 ablation resulted in accumulation of CD8 ISP cells. However, experiments presented in this article revealed that the number of ISPs was not increased in the Tpl2−/− mice and suggested that T cell lymphomas arising in these mice may derive from chronically stimulated mature peripheral T cells. This leaves us with the puzzling question of why transgene-negative Tpl2−/− mice do not develop T cell lymphomas. Although we do not have a definitive answer to this question, we speculate that responding to an array of TCR ligands that are heterogeneous with regard to their TCR affinities may give rise to compensatory mechanisms that prevent oncogenesis. Responding to heterogeneous ligands, for example, may allow the development of tumor-specific CD8 SP cytotoxic T cells that interfere with tumor induction and/or progression. We should add that our data are in agreement with the results of earlier studies that had shown that TCR transgenes increase the susceptibility of thymocytes to malignant transformation (32).

CTLA4−/− mice exhibit a dramatic expansion of the CD4 SP T cells, and they die within 3–4 weeks with massive lymphocytic infiltrates in nonlymphoid organs (33, 34). This phenotype is abrogated by crossing the TCR2C transgene into the CTLA4−/− genetic background. TCR2Ctg/tg/CTLA4−/− mice exhibit normal positive and negative thymocyte selection and normal Treg development. Moreover, CD8 SP T cells from these mice exhibit a normal primary response but enhanced secondary response to TCR stimulation (33). Therefore, there are significant similarities between the TCR2Ctg/tg/Tpl2−/− and the TCR2Ctg/tg/CTLA4−/− mice, as expected from our data. In addition to the similarities, however, there may also be differences between these mice. One such difference may be the development of CD8 SP T cell lymphomas in the TCR2Ctg/tg/Tpl2−/− mice, which has not been described in the TCR2Ctg/tg/CTLA4−/− mice. We propose that the differences may be because CTLA4 and its ligands may be subject to different regulatory influences in different cell types. As a result, it is not surprising that the Tpl2 ablation, which may affect differently the regulation of CTLA4 and its ligands in different cell types, may also exhibit an organismal phenotype that is not identical to the organismal phenotype of CTLA4 ablation.

The observation that inhibition of Tpl2 enhances the response of T cells to antigen both in culture and in animals may have significant translational implications. Earlier studies had indeed shown that treatment of cancer patients with ipilimumab, an anti-CTLA4 antibody, inhibits tumor growth. However, the complete block of CTLA4 with ipilimumab was toxic, and the patients developed severe dermatitis, enterocolitis, hypophysitis, uveitis, hepatitis, and nephritis (35). Because inhibition of Tpl2 blocks the induction of CTLA4 only partially, Tpl2 inhibitors when available, may have a therapeutic effect in the absence of severe toxicity. The facts that TCR2Ctg/tg/Tpl2−/− mice did not exhibit severe short-term toxicity and that Tpl2−/− mice do not develop autoimmunity support this suggestion.

Materials and Methods

Mice.

Tpl2−/− mice in the C57BL/6 background were generated as described in ref. 36. Brother–sister mating of TCR2Ctg/tg/Tpl2−/− mice, derived from crossing the TCR2C transgene into the Tpl2−/− genetic background, gave rise to TCR2Ctg/tg/Tpl2−/−, TCR2Ctg/tg/Tpl2+/−, or TCR2Ctg/tg/Tpl2+/+ experimental mice.

Antibody Staining of Thymocytes and Splenocytes and Flow-Cytometric (FACS) Analysis.

Single-cell pellets of mouse splenocytes and thymocytes were resuspended in a hypotonic red blood cell (RBC) lysis buffer containing 0.165 M NH4Cl2. Cells were stained for 20 min with fluorescent marker-conjugated antibodies and analyzed as described in the SI Materials and Methods.

Splenocyte Cultures.

Single-cell suspensions of spleen cells were cultured at 37°C in an atmosphere of 5% CO2 in RPMI medium. In some of the experiments, total splenocyte cultures (5 × 106 cells/ml) were stimulated for 24 h with 4 μg/ml ConA (Sigma) and then cultured in the presence of recombinant IL-2 (Invitrogen) for an additional 3–5 days. Further details are provided in SI Materials and Methods.

Isolation of CD3+ or CD8+ T Cells.

CD3+ or CD8+ T cells were purified by using reverse selection columns and following the instructions of the manufacturer (R&D). Briefly, 109 cells were loaded onto CD3+ or CD8+ T cell separation columns. After washing of the columns, cells were eluted and cultured in RPMI for 3 h. Cultured cells were stimulated as described in Results.

Cell Proliferation Assays.

[3H]Thymidine incorporation assay.

We stimulated 100-μl aliquots of total splenocytes (106 cells/ml) with soluble anti-CD3, anti-CD3 plus anti-CD28, the peptide EQYKFYSV (dEV8), or the peptide SIYRYYGL (SIY), and [3H]thymidine incorporation was measured as described in SI Materials and Methods.

CFSE assay.

Total splenocytes were labeled with CFSE (Molecular Probes) (for details see SI Materials and Methods). To measure the dilution of CFSE in CD8 SP splenocytes over time, cells were harvested at sequential time points, stained with PE-conjugated anti-CD8, and analyzed by flow cytometry (FACS-Calibur, (Beckton Dickinson).

Western Blot Analysis.

Western blotting was performed as described in refs. 24 and 36. Details on the method and the antibodies are provided in SI Materials and Methods.

Immunohistochemistry.

Immunohistochemistry was performed on paraffin-embedded tissue sections (4–6 μm), as described in SI Materials and Methods.

Supplementary Material

Supporting Information
pnas_0708381104_index.html (12.2KB, html)

ACKNOWLEDGMENTS.

We thank D. Kontoyiannis, V. Panoutsakopoulou, and C. Mamalaki for stimulating discussions. This work was supported by Grants AICR07–0072 and UICC-ICRETT-969 (to C.T.) and R01 CA095431 (to P.N.T.).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0708381104/DC1.

References

  • 1.van der Merwe PA, Davis SJ. Molecular interactions mediating T cell antigen recognition. Annu Rev Immunol. 2003;21:659–684. doi: 10.1146/annurev.immunol.21.120601.141036. [DOI] [PubMed] [Google Scholar]
  • 2.Mitchison NA. T-cell–B-cell cooperation. Nat Rev Immunol. 2004;4:308–312. doi: 10.1038/nri1334. [DOI] [PubMed] [Google Scholar]
  • 3.Greenwald RJ, Freeman GJ, Sharpe AH. The B7 family revisited. Annu Rev Immunol. 2005;23:515–548. doi: 10.1146/annurev.immunol.23.021704.115611. [DOI] [PubMed] [Google Scholar]
  • 4.Wang J, Stohlman SA, Dennert G. TCR cross-linking induces CTL death via internal action of TNF. J Immunol. 1994;152:3824–3832. [PubMed] [Google Scholar]
  • 5.Wahl SM, et al. Bacterial cell wall-induced immunosuppression: Role of transforming growth factor β. J Exp Med. 1988;168:1403–1417. doi: 10.1084/jem.168.4.1403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Lin L, Spoor MS, Gerth AJ, Brody SL, Peng SL. Modulation of Th1 activation and inflammation by the NF-κB repressor Foxj1. Science. 2004;303:1017–1020. doi: 10.1126/science.1093889. [DOI] [PubMed] [Google Scholar]
  • 7.Brunkow ME, et al. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet. 2001;27:68–73. doi: 10.1038/83784. [DOI] [PubMed] [Google Scholar]
  • 8.Kuo CT, Veselits ML, Leiden JM. LKLF: A transcriptional regulator of single-positive T cell quiescence and survival. Science. 1997;277:1986–1990. doi: 10.1126/science.277.5334.1986. [DOI] [PubMed] [Google Scholar]
  • 9.Chiang YJ, et al. Cbl-b regulates the CD28 dependence of T cell activation. Nature. 2000;403:216–220. doi: 10.1038/35003235. [DOI] [PubMed] [Google Scholar]
  • 10.Anandasabapathy N, et al. GRAIL: An E3 ubiquitin ligase that inhibits cytokine gene transcription is expressed in anergic CD4+ T cells. Immunity. 2003;18:535–547. doi: 10.1016/s1074-7613(03)00084-0. [DOI] [PubMed] [Google Scholar]
  • 11.McGargill MA, Wen BG, Walsh CM, Hedrick SM. A deficiency in Drak2 results in a T cell hypersensitivity and an unexpected resistance to autoimmunity. Immunity. 2004;21:781–791. doi: 10.1016/j.immuni.2004.10.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Riley JL, June CH. The CD28 family: A T cell rheostat for therapeutic control of T cell activation. Blood. 2005;105:13–21. doi: 10.1182/blood-2004-04-1596. [DOI] [PubMed] [Google Scholar]
  • 13.Teft WA, Kirchhof MG, Madrenas J. A molecular perspective of CTLA-4 function. Annu Rev Immunol. 2006;24:65–97. doi: 10.1146/annurev.immunol.24.021605.090535. [DOI] [PubMed] [Google Scholar]
  • 14.Baroja ML, et al. Inhibition of CTLA-4 function by the regulatory subunit of serine/threonine phosphatase 2A. J Immunol. 2002;168:5070–5078. doi: 10.4049/jimmunol.168.10.5070. [DOI] [PubMed] [Google Scholar]
  • 15.Chuang E, et al. The CD28 and CTLA-4 receptors associate with the serine/threonine phosphatase PP2A. Immunity. 2000;13:313–322. doi: 10.1016/s1074-7613(00)00031-5. [DOI] [PubMed] [Google Scholar]
  • 16.Chikuma S, Imboden JB, Bluestone JA. Negative regulation of T cell receptor-lipid raft interaction by cytotoxic T lymphocyte-associated antigen 4. J Exp Med. 2003;197:129–135. doi: 10.1084/jem.20021646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Gomes NA, Gattass CR, Barreto-De-Souza V, Wilson ME, DosReis GA. TGF-β mediates CTLA-4 suppression of cellular immunity in murine kalaazar. J Immunol. 2000;164:2001–2008. doi: 10.4049/jimmunol.164.4.2001. [DOI] [PubMed] [Google Scholar]
  • 18.Paust S, Lu L, McCarty N, Cantor H. Engagement of B7 on effector T cells by regulatory T cells prevents autoimmune disease. Proc Natl Acad Sci USA. 2004;101:10398–10403. doi: 10.1073/pnas.0403342101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Grohmann U, et al. CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat Immunol. 2002;3:1097–1101. doi: 10.1038/ni846. [DOI] [PubMed] [Google Scholar]
  • 20.Patriotis C, Makris A, Bear SE, Tsichlis PN. Tumor progression locus 2 (Tpl-2) encodes a protein kinase involved in the progression of rodent T-cell lymphomas and in T-cell activation. Proc Natl Acad Sci USA. 1993;90:2251–2255. doi: 10.1073/pnas.90.6.2251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Makris A, Patriotis C, Bear SE, Tsichlis PN. Genomic organization and expression of Tpl-2 in normal cells and Moloney murine leukemia virus-induced rat T cell lymphomas: Activation by provirus insertion. J Virol. 1993;67:4283–4289. doi: 10.1128/jvi.67.7.4283-4289.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Patriotis C, Makris A, Chernoff J, Tsichlis PN. Tpl-2 acts in concert with Ras and Raf-1 to activate mitogen-activated protein kinase. Proc Natl Acad Sci USA. 1994;91:9755–9759. doi: 10.1073/pnas.91.21.9755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Ceci JD, et al. Tpl-2 is an oncogenic kinase that is activated by carboxy-terminal truncation. Genes Dev. 1997;11:688–700. doi: 10.1101/gad.11.6.688. [DOI] [PubMed] [Google Scholar]
  • 24.Tsatsanis C, Patriotis C, Tsichlis PN. Tpl-2 induces IL-2 expression in T cell lines by triggering multiple signaling pathways that activate NFAT and NF-κB. Oncogene. 1998;17:2609–2618. doi: 10.1038/sj.onc.1202460. [DOI] [PubMed] [Google Scholar]
  • 25.Tsatsanis C, Patriotis C, Bear SE, Tsichlis PN. The Tpl-2 protooncoprotein activates the nuclear factor of activated T cells and induces interleukin 2 expression in T cell lines. Proc Natl Acad Sci USA. 1998;95:3827–3832. doi: 10.1073/pnas.95.7.3827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Sha WC, et al. Selective expression of an antigen receptor on CD8-bearing T lymphocytes in transgenic mice. Nature. 1988;335:271–274. doi: 10.1038/335271a0. [DOI] [PubMed] [Google Scholar]
  • 27.Sykulev Y, Vugmeyster Y, Brunmark A, Ploegh HL, Eisen HN. Peptide antagonism and T cell receptor interactions with peptide–MHC complexes. Immunity. 1998;9:475–483. doi: 10.1016/s1074-7613(00)80631-7. [DOI] [PubMed] [Google Scholar]
  • 28.Vaillant F, Blyth K, Andrew L, Neil JC, Cameron ER. Enforced expression of Runx2 perturbs T cell development at a stage coincident with β-selection. J Immunol. 2002;169:2866–2874. doi: 10.4049/jimmunol.169.6.2866. [DOI] [PubMed] [Google Scholar]
  • 29.Garcia KC, et al. Structural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen. Science. 1998;279:1166–1172. doi: 10.1126/science.279.5354.1166. [DOI] [PubMed] [Google Scholar]
  • 30.Christoforidou AV, Papadaki HA, Margioris AN, Eliopoulos GD, Tsatsanis C. Expression of the Tpl2/Cot oncogene in human T cell neoplasias. Mol Cancer. 2004;3:34. doi: 10.1186/1476-4598-3-34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Babu G, Waterfield M, Chang M, Wu X, Sun SC. Deregulated activation of oncoprotein kinase Tpl2/Cot in HTLV-I-transformed T cells. J Biol Chem. 2006;281:14041–14047. doi: 10.1074/jbc.M512375200. [DOI] [PubMed] [Google Scholar]
  • 32.Kelly JA, et al. Stat5 synergizes with T cell receptor/antigen stimulation in the development of lymphoblastic lymphoma. J Exp Med. 2003;198:79–89. doi: 10.1084/jem.20021548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Chambers CA, Sullivan TJ, Truong T, Allison JP. Secondary but not primary T cell responses are enhanced in CTLA-4-deficient CD8+ T cells. Eur J Immunol. 1998;28:3137–3143. doi: 10.1002/(SICI)1521-4141(199810)28:10<3137::AID-IMMU3137>3.0.CO;2-X. [DOI] [PubMed] [Google Scholar]
  • 34.Waterhouse P, Bachmann MF, Penninger JM, Ohashi PS, Mak TW. Normal thymic selection, normal viability and decreased lymphoproliferation in T cell receptor-transgenic CTLA-4-deficient mice. Eur J Immunol. 1997;27:1887–1892. doi: 10.1002/eji.1830270811. [DOI] [PubMed] [Google Scholar]
  • 35.Beck KE, et al. Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4. J Clin Oncol. 2006;24:2283–2289. doi: 10.1200/JCO.2005.04.5716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Dumitru CD, et al. TNF-α induction by LPS is regulated posttranscriptionally via a Tpl2/ERK-dependent pathway. Cell. 2000;103:1071–1083. doi: 10.1016/s0092-8674(00)00210-5. [DOI] [PubMed] [Google Scholar]

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