Pten loss in CD4 T cells enhances their helper function but does not lead to autoimmunity or lymphoma

Abstract

Pten, one of the most common tumor suppressors in human cancers, antagonizes signaling by the PI3K pathway. Mice with thymocyte-specific deletion of Pten rapidly developed peripheral lymphomas and autoimmunity, which may have been due to failed negative selection of thymocytes or from dysregulation of post-thymic T cells. We induced conditional deletion of Pten from CD4 helper T cells using a Cre knocked into the Tnfrsf4 (OX40) locus to generate OX40^CrePten^f mice. Pten-deficient helper T cells proliferated more and produced higher concentrations of cytokines. The OX40^CrePten^f mice had a general increase in the number of lymphocytes in the lymph nodes, but not in the spleen. When transferred into wild-type mice, Pten-deficient helper T cells enhanced anti-Listeria responses and the clearance of tumors under conditions in which wild-type T cells had no effect. Moreover, inflammatory responses were exaggerated and resolved later than in OX40^CrePten^f mice than in wild type mice. However, in contrast to models of thymocyte-specific Pten deletion, lymphomas and autoimmunity were not observed, even in older OX40^CrePten^f mice. Hence, loss of Pten enhances helper T cell function without obvious deleterious effects.

Introduction

CD4 T cells support the coordinated activation of other leukocytes during immune responses. For instance, CD4 T cells secrete inflammatory cytokines during contact hypersensitivity (CHS) reactions, augment tumor surveillance, help B cells during the germinal center reactions, and license dendritic cells to express high levels of MHC and costimulatory ligands (1-4). Full activation of naive CD4 T cells requires persistent stimulation of the TCR and CD28 over a period of about 24 hours. During this time, the T cells interact with antigen-presenting cells (APCs) and integrate signals needed to increase metabolism, grow in size and upregulate cytokine, chemokine and costimulatory receptors (5). After initial activation, CD4 T cells begin to divide in response to cytokines and express additional costimulatory receptors such as ICOS and OX40. They then differentiate into different T helper (Th) cell lineages which secrete cytokines such as interferon-γ (IFNγ), interleukin 4 (IL-4) or IL-17. Eventually, most CD4 Th cells die through apoptosis, but some survive as CD4 memory Th cells (6). Similarly, after initial activation, naïve CD8 T cells differentiate to become CTLs and the ones that survive the cycle of expansion and contraction become CD8 memory T cells (7).

The TCR and many costimulatory and cytokine receptors activate phosphoinositide 3-kinases (PI3Ks) (8). The class I PI3Ks (p110α, p110β, p110γ and p110δ) use PtdIns(4,5)P₂ as their preferred substrate to generate the second messenger molecule PtdIns(3,4,5)P₃, which helps activate PH domain containing proteins such as Akt. Akt phosphorylates Foxo transcription factors and proteins associated with mTOR complex activation (9, 10). PI3K activity is also required for optimal Erk phosphorylation in T cells (11). By these and other mechanisms, PI3Ks contribute to the proliferation, growth, survival, cytokine production, trafficking, and homeostasis of CD4 T cells (8-16). In T cells, roles for p110α and p110β have not been reported, but the p110γ isoform is activated by G-protein coupled receptors and regulates basal motility in the lymph node (LN), chemotaxis of effector T cells to sites of inflammation and the survival of memory T cells (17-21). p110γ can also regulate chemotaxis in human T cells (22). p110δ is the functionally dominant isoform downstream of TCR, ICOS and the IL-2 receptors and controls Ag-specific events such as differentiation (11, 12, 23-25). PI3K activity remains high for several days after CD4 T cell activation (26), and using acute inhibition with an isoform-selective inhibitor, we demonstrated that p110δ activity is required beyond the first 24 hours following TCR activation to regulate cytokine production (16). We also demonstrated the p110δ is a major regulator of cytokine production in human T cells from healthy, atopic and arthritic individuals (16). Given the importance of Th cytokine production in supporting protective and pathological immune responses, it is important not only to understand how the PI3K pathway is activated, but also how it is sustained or curtailed.

Pten de-phosphorylates PtdIns(3,4,5)P₃ to produce PtdIns(4,5)P₂, thus terminating PI3K signaling. Pten is frequently lost or mutated in human cancers, including 20% of T cell acute lymphoblastic leukemias (27). Pten expression is also lost in commonly used human T cell lines such as Jurkat which may have confounded some studies where these cells are used to study T cell signaling (28, 29). Conditional deletion of Pten in thymocytes led to defective negative selection, progressive lymphoproliferation, autoimmunity and CD4 T cell lymphomas (30-32). Similar effects were observed in mice that over-expressed micro-RNAs that target Pten (33). After activation, peripheral Pten^−/− T cells proliferated more, resisted apoptosis, and failed to contract after superantigen stimulation (30, 34). Pten^−/− T cells also could be activated in the absence of CD28 co-stimulation and resisted CTLA4-Ig-induced anergy induction and so Pten was suggested to impose a threshold for activation during initial TCR sensing (32). Although Pten expression levels may modulate immediate responses downstream of the TCR in thymocytes and naïve T cells, the role of Pten during an ongoing immune response is not well understood.

To address the role of Pten in mature CD4 Th cells, we conditionally deleted the Pten gene in Th cells using a Cre-recombinase gene knocked into Tnfrsf4 (the gene that encodes OX40) (35). OX40 is transiently expressed about 24 hours after activation in the majority of CD4 T cells, but rarely in CD8 T cells (36). OX40 is also constitutively expressed on regulatory T cells (Tregs) (36). We found that deletion of Pten after TCR stimulation regulated the magnitude and duration of T cell responses, but not apoptosis or contraction. Furthermore, contrary to when Pten was deleted in thymocytes, lymphoma did not develop when the deletion occurred in activated Th cells. Instead, over-production of cytokines in these mice leads to altered homeostasis of the lymphocyte compartment, and enhanced inflammatory, anti-bacterial and anti-tumor responses.

Materials and Methods

Mice

All mice were maintained under specific pathogen-free conditions. All experiments were performed in accordance with U.K. Home Office regulations. OT2 (37), Rag1^−/− (38), Rag2^−/− (39), OX40^Cre (35), Pten^f (40) and R26^YFP (41) and R26^tdRFP (42) mice were described previously

Reagents

Unless otherwise stated, all chemicals were from Sigma-Aldrich. IC87114 (IC)³ was synthesized as previously described (16) and anti-CD3 (2C11) was purified in house. IL-2 was synthesized by GSK. Cells were cultured in RPMI 1640 supplemented with 100 U/ml penicillin/streptomycin, 2 mM L-glutamine, 10 mM HEPES, 20 μM 2-ME, and 5% FCS at 5% CO2.

Antibodies, flow cytometry and Western blot

All Abs were from eBioscience except for Fas and CD4-V500 (BD); Pten, p-Akt and p-Erk (Cell Signal Technologies; and anti-rabbit Ig Alexa 647 (Invitrogen). To stain for transcription factors, Pten or intracellular cytokine, FoxP3 staining buffer set or IC Fixation Buffer Kits (both eBioscience) were used, respectively. To detect signaling molecules, CD4 T cells were purified with a Naïve CD4 T cell Kit (Miltenyi) and activated with 1μg/mL anti-CD3 on irradiated APCs. Every 24 hr, an aliquot of cells were treated with Fix Buffer I and Permeabilization Buffer 3 (BD) according to manufacturer’s instruction, and cells stained at the end of the time course. Apoptotic cells were detected with 7-AAD +/− Annexin V (BD), according to manufacturer’s instructions. Flow cytometry data was acquired with FACSCalibur or LSRII instruments (BD) and analyzed with FlowJo (Tree Star). Cells were sorted using a FACSAria (BD) machine. Cell counts were performed either using a CASYCounter or FlowCount Fluorospheres (Beckman Coulter).

Cell purification, proliferation and cytokine assays

CD4 T cells were purified, stimulated, and proliferation and cytokine production measured as described previously (12, 16). To measure cytokines by intracellular FACS or by Mouse Cytokine Array Panel A (R&D Systems), cells were stimulated with 1μg/mL PdBu and 5nM Ionomycin for 5 hrs and 10μg/mL Brefeldin A was added for the final 2hrs. For the Mouse Cytokine Array Panel A, lysates then were made from 5×10⁷ cells and cytokines detected according to manufacturer’s instructions. Pixel density was analyzed using Aida Image Analysis software.

T_reg suppression assay

Suppression assays with anti-CD3-coated APCs were performed as described previously (43). Suppression was calculated as the amount of proliferation when Tregs were present compared to when they were absent. (% suppression = (cpm responders with Tregs/cpm responders alone) * 100).

Autoantibody detection

Autoantibodies were detected using a Hep2 ANA kit (The Binding Site). MRL positive control serum was a gift from L. Martensson-Bopp.

Transwell assays

OT2 Pten^f and OT2 OX40^CrePten^f cells were activated in vitro with OVA peptide for 3 days, and then live CD4 T cells were purified. Next, CD4 T cells were co-cultured at various ratios with CD45.1⁺ naïve WT splenocytes, either in direct contact or separated by 0.4μM Transwell inserts (Fisher). After three days, the numbers of CD45.1⁺ CD4, CD8 and B220 cells were calculated.

CHS assays

CHS assays were done as described previously (16). Briefly, trinitrochlorobenzene (TNCB⁴)-sensitized mice were re-challenged with TNCB and dosed twice daily with 30mg/kg IC or 1% methylcellulose vehicle control for 2 days. 26 days after the first challenge, the same ear was re-challenged to examine secondary responses. These mice had not received drug during the primary elicitation. Ear size was measured with a micrometer (Kroeplin).

Adoptive transfer assays

CD45.1 hosts were injected i.v. with 10⁶ OT2 Pten^f or OT2 OX40^CrePten^f CD4 T cells and the next day injected s.c. with PBS or 50 μg LPS O26:B6+ 1mg OVA. Skin draining LNs were harvested on day 3, 6, or 9 after immunization.

Listeria monocytogenes (Lm⁵) assays

CD45 mis-matched hosts were injected i.v. with PBS or 1.5×10⁶ OT2 Rag2^−/− or OT2 OX40^CrePten^fRag2^−/− cells. The following day, hosts were i.v. injected with 10⁷ CFU of attenuated OVA-secreting LM (ActA-LM-OVA)(44). After 3 days, splenocytes were stained for intracellular cytokines. APC-labeled SIINFEKL-MHC Class I tetramer (Beckman-Coulter) were used to detect OVA-specific CD8 T cells.

Tumor assays

CD45.1⁺ hosts were injected i.v. with PBS or 2×10⁵ CD45.2⁺ OT2 Pten^f Rag2^−/− or OT2 OX40^CrePten^fRag2^−/− cells. The following day, 5×10⁵ EG7 were injected s.c. into hosts. Tumors were palpable between days 10-11. On day 14 after tumor injection, mice were culled and tumors were weighed.

Statistics

Statistics were calculated with GraphPad or SSCS software. The following symbols are used on graphs and tables: * 0.05≥p>0.01 **0.01≥p>0.001 ***0.001≥p

Results

PI3K signaling in OX40^CrePten^f T cells is sustained

In OX40^Cre mice, Cre is expressed almost exclusively in activated CD4 T cells and Tregs and only 2-5% of CD8 T cells (35). Using reporter mice, we detected Cre activity in 20% of CD4⁺ T cells one day after activation, increasing to 80% after three days (Fig S1A). PCR analysis revealed nearly complete recombination of the Pten locus in YFP⁺, OX40^CrePten^f CD4 T cells whereas no deletion was observed in YFP⁻ cells. This showed that the YFP accurately reported Cre activity on the Pten^f gene (Fig S1B). Flow cytometry showed loss of Pten protein in OX40^CrePten^f CD4⁺CD25⁺ and CD4⁺CD44⁺ T cells, but not in CD4⁺CD44⁻ T cells (Fig. 1A). These results confirm that Pten was deleted in Treg and memory T cells, but not in naïve T cells. When stimulated with anti-CD3 and APCs, Akt and Erk phosphorylation were sustained for longer in OX40^CrePten^f CD4 T cells compared to controls (Fig. 1B). This was consistent with the kinetics observed for Cre expression after activation (Fig S1A)

Figure 1. Loss of Pten leads to altered signalling and LN hyperplasia.

A) Pten was detected ex vivo by intracellular flow cytometry in CD25⁺, CD44^lo and CD44^hi CD4 T cells mice. n≥5 for each genotype. p values were determined with Student’s t-test. B) CD62L⁺CD4⁺ T cells were activated by irradiated APCs and anti-CD3, and the level of Akt and Erk phosphorylation were measured at 24 hr intervals. Representative histograms and graphs of median fluorescence intensities are on the right. Data shows mean±SD for three mice from each genotype, and represents one of three independent experiments. Repeated measures ANOVA shows statistical significance over the whole time course of 0.05≥p≥0.01 for both phospho-Akt and phospho-Erk. The results of Bonferroni post-tests also showed that phospho-Akt was strongly increased in OX40^CrePten^f cells at day three. C,D) Blood, spleen, and LN cells from 5-10 week old Pten^f and OX40^CrePten^f mice were stained for lineage and activation markers, and analyzed by flow cytometry. C) Representative histograms of activation markers in CD4 cells. n≥5 for each genotype. D) Total numbers of B cells and CD4 and CD8 T cells. Student’s t test was used to calculate p values.

Pten regulates lymphocyte homeostasis

Pten deletion led to enhanced CD4 T cell activation in non-challenged animals. Ultimately, this led to enhanced activation of CD4 T cells as evidenced by increased proportion of cells expressing high levels of CD44 and lower levels of CD62L, and CD45RB in LNs, spleen and blood of OX40^CrePten^f mice (Fig. 1C). However, unlike mice with Pten deletion in thymocytes (30), auto-antibodies were not detected in OX40^CrePten^f mice even at an advanced age (Fig. S2). Furthermore, histology of 22 organs from 19 week old OX40^CrePten^f mice (n=5) and controls (n=5) showed no signs of inflammation or autoimmunity. More than 80% of mice with a germ-line heterozygous deletion in Pten form lymphomas by 24 weeks of age and 100% of mice with a thymocyte deletion of Pten develop lymphomas by 12 weeks of age (30, 45, 46). These lymphomas derive from the thymus (47-49), but this did not exclude the possibility of lymphomas also arising in mice with post-thymic deletion of Pten. However, none of 31 OX40^CrePten^f mice culled between 25-37 weeks of age showed evidence of lymphomas. In addition, none of five mice aged over 52 weeks had lymphomas. Therefore the pathology associated with Pten deletion in all T cells was not evident when using OX40^Cre. Although OX40^CrePten^f mice did not develop lymphomas, their LNs –but not spleens –were enlarged from five weeks after birth with 2-3-fold more T and B cells in the LNs compared to littermates. In contrast, lymphocyte numbers in the spleen and blood were similar (Fig. 1D).

Since OX40^cre is expressed by regulatory T cells in the thymus (35), and because Pten can affect Treg expansion in vitro (50), we enumerated Tregs in OX40^CrePten^f mice. There were similar numbers of Tregs in the thymi of OX40^CrePten^f mice, but more in the LNs (Fig. S3A-D). Consistent with previous results (50, 51), there were no significant differences in suppression of wild type (WT) responders by OX40^CrePten^f Tregs or in the ability of Tregs of either genotype to suppress OX40^CrePten^f responders (Fig. S3E). Hence, deficiencies in Tregs or susceptibility of Th cells to suppression are unlikely to have caused LN hyperplasia in OX40^CrePten^f mice.

Lymphocyte expansion is controlled in trans by Th cells

We next considered whether Pten-deficient Th cells altered homeostasis of Pten-sufficient bystander lymphocytes. To test this hypothesis, we generated mixed bone marrow chimeras in lethally irradiated Rag^−/− mice, in which 50% of the donor bone marrow derived from CD45.1⁺ WT SJL mice and 50% derived from either CD45.2⁺ OX40^CrePten^f or CD45.2⁺ WT mice. Eight weeks after reconstitution, the number of lymphocytes in spleens of hosts receiving WT:SJL or OX40^CrePten^f:SJL bone marrow was similar (Fig. 2A). By contrast, there was 3-4-fold more B and T cells in LNs of mice receiving SJL:OX40^CrePten^f bone marrow than controls (Fig. 2B). When the ratio of CD45.1 to CD45.2 cells was analyzed, however, we found both donors contributed equally to cells re-populating the LNs and spleens, suggesting that OX40^CrePten^f CD4 T cells support general accumulation of LN cells independently of their Pten status.

Figure 2. OX40^CrePten^f CD4 T cells support expansion of lymphocytes.

Lethally irradiated Rag2^−/− were reconstituted with a 50:50 mix of CD45.1⁺ WT SJL BM and either CD45.2⁺ WT (n=7) or OX40^CrePten^f (n=9) bone marrow. A) Spleen and B) LN were analyzed eight weeks later for the number of lymphocytes and the relative contribution (ratio) of CD45.1 and CD45.2 bone marrow. Students t test was used to calculate p values. C) Activated CD4 cells from CD45.2⁺ OT2 Pten^f (n=3) and OT2 OX40^CrePten^f (n=3) mice were co-cultured at various ratios with CD45.1⁺ splenocytes while in direct contact (top panels) or while separated by a Transwell filter (bottom panels). After three days, the number of CD45.1⁺ lymphocytes was calculated. Data shows mean+SD. Three-way ANOVA was used to calculate statistical significance in number of responder cells recovered. OT2 OX40^CrePten^f vs.WT OT2 without Transwell: CD4 and CD8 T cells 0.05≥p≥0.01; B cells p≤0.001. No Transwell vs. Transwell: No significant difference in either OT2 OX40^CrePten^f or WT OT2. Results of significant Bonferroni post-tests for each titration point are indicated on the graph.

To test if a soluble factor was needed to expand lymphocytes, Pten^f or OT2 OX40^CrePten^f CD4 OT2 T cells were activated using the OVA-derived peptide recognized by the OT2 TCR. The activated OT2 T cells were then purified and co-cultured with unfractionated WT lymphocytes. After three days, the number of WT lymphocytes was counted (Fig. 2C). OT2 OX40^CrePten^f CD4 T cells maintained WT lymphocytes better than controls when effectors were in direct contact with responders . This difference between genotypes was unaffected by inclusion of a Transwell filter, demonstrating that expansion of responders was due to secretion of a soluble factor by OX40^CrePten^f effector cells.

Pten regulates cytokine production by activated CD4 T cells

In an effort to determine which cytokines might be responsible for the hyperplasia, LN cells were stimulated with PdBu and ionomycin to stimulate cytokine production. Next, lysates made from equal numbers of cells were used to probe a cytokine array testing 40 cytokines and chemokines and the signal from OX40^CrePten^f cells was divided by the signal from control cells (Fig. S4A). This chemiluminescent array is a relative measure of cytokine production, where a small difference in pixel density between genotypes can indicate a large difference in the absolute amount of cytokine. Although we could not identify a single factor responsible for hyperplasia, we found a limited range of factors were over-produced in OX40^CrePten^f LN, and these included cytokines made by different Th subsets and those that could be induced indirectly by Th cells, such as the chemokine CXCL9 (also known as MIG: Monokine-induced by IFNγ). Hence, we postulate that an environment rich in multiple helper T cell-dependent cytokines resulted in LN hyperplasia.

OT2⁺OX40^CrePten^f showed enhanced proliferation and produced greater amounts of IL-2, IL-4 and, IFNγ when stimulated with peptide (Fig. S4C-E). The enhanced proliferation and cytokine production could be blocked by inhibitors against p110δ, Akt and Erk added 24h after activation (Fig. S4B-E). The results from figure S4 could be affected the increased number of CD4⁺ T cells with an activated phenotype (32). We therefore purified naïve CD62L⁺ and antigen-experienced CD62L⁻ CD4 T cells from OX40^CrePten^f mice. Both populations showed a 2-3 fold increase in proliferation and cytokine production when stimulated with anti-CD3 (Fig. 3A-D). High concentrations of the pan-PI3K inhibitor LY294002 (LY) or the p110δ-selective inhibitor IC87114 (IC) reduced proliferation and cytokine production in OX40^CrePten^f and WT cells to near background levels. At lower concentrations of IC, OX40^CrePten^f T cells were less sensitive than WT T cells, suggesting that the activity of PI3K isoforms other than p110δ are also normally restrained by Pten (Fig. 3A-D). To examine Th cell differentiation, CD4 T cells were activated in vitro in presence of IL-12 to produce IFNγ-secreting Th1 cells. A greater proportion of OT2 OX40^CrePten^f T cells had divided and produced IFNγ than controls (Fig 3E-G). In addition, IFNγ⁺ OX40^CrePten^f T cells stained more brightly for IFNγ than did WT cells (Fig. 3E,H). Together these data demonstrate that Pten acts to restrain the number of cells dividing, the number of those cells producing cytokine, and the amount of cytokine produced by individual cells.

Figure 3. Pten antagonizes p110δ-regulated proliferation and cytokine production.

A-D) CD4 T cells from Pten^f (n=3) and OX40^CrePten^f (n=3) were sorted into A,C) CD62L⁺ and B,D) CD62L⁻ populations and stimulated with anti-CD3 and either DMSO, 10μM LY, 10μM, 1μM or 0.1μM IC (doses represented by triangle). Two days later, A, B) proliferation and C,D) IFNγ production were analyzed. Data shows mean± SEM. Data represents three independent experiments. E-H) CFSE-labelled OT2 Pten^f and OT2 OX40^CrePten^f were stimulated with peptide in Th1-skewed conditions, and cells were stained after three days for intracellular IFNγ. Data is representative of three independent experiments. E) Representative plots. F) CFSE dilution was used to determine the number of mitoses/generations each cell had gone through. G) The proportion of IFNγ⁺ cells was determined for each generation. H) The MFI in IFNγ⁺ cells was determined for each generation to quantify the amount of IFNγ each cell was making.

Pten regulates the magnitude and kinetics of immune responses

To understand if altered homeostasis caused by Pten-deficient Th cells affected immune responses, we used a CHS model where increased ear thickness can be used as a measure of T cell-dependent inflammation. Mice were sensitized by application of TNCB on their abdomens and then re-challenged six and 32 days later on their ear to elicit primary and secondary hypersensitivity responses, respectively. Mice were dosed orally with either IC or vehicle control during re-challenges (Fig. 4A). The magnitude and duration of the primary response was greater in OX40^CrePten^f than in WT mice. The enhanced inflammation was partially p110δ dependent because IC reduced ear swelling in WT and OX40^CrePten^f mice. The magnitude and duration of the secondary responses was also greater in OX40^CrePten^f than WT mice (Fig. 4B). IC again reduced ear swelling in both groups. We conclude that OX40^CrePten^fl mice experience a greater inflammatory immune response which nonetheless can be resolved and which can be attenuated by oral administration of p110δ inhibitors.

Figure 4. Pten limits CHS responses by a p110δ-dependent mechanism.

A) TNCB-sensitized WT and OX40^CrePten^f mice were challenged with TNCB on an ear. Mice were dosed with 30mg/kg IC or vehicle control one hr before challenge and twice daily for two days. Ear size was measured before challenge and at 24 hr intervals. B) TNCB-sensitized WT and OX40^CrePten^f mice were challenged with TNCB on an ear and then re-challenged on the same ear 24 days later. Mice were also dosed with 30mg/kg or vehicle control and then twice daily for two days during the secondary response only. Data shows mean±SD of ≥6 mice for each group, and represents two experiments for un-drugged mice and one experiment for IC-drugged mice. C) Statistics were measured by repeated measures ANOVA. Table shows results of Bonferroni post-tests on each day of ear measurement.

In order to follow the activation kinetics of OX40^CrePten^f T cells in vivo, we adoptively transferred OT2 Pten^f or OX40^CrePten^f CD4 OT2 T cells into WT hosts. Following immunization with LPS and OVA protein, there were significantly more OT2 OX40^CrePten^f donor cells than controls after six days of activation. However, nine days after activation both donor cell types had returned to baseline levels (Fig. 5A). This shows that Pten can control the magnitude of T cell response to Ag after initial TCR activation events, yet the lack of Pten expression does not interfere with the contraction of the T cell response. Consistent with this, we found no difference in apoptosis of OT2 OX40^CrePten^f cells compared to controls during the first 72 h following activation in vitro (Fig. 5B). Activated OT2 OX40^CrePten^f had a mild survival advantage in response to anti-Fas-induced death, but behaved similarly to controls in response to cytokine deprivation, anti-CD3 stimulation, and γ-irradiation (Fig. 5C). Hence, while activation is enhanced, the absence of Pten does not necessarily interfere with apoptotic signaling in OX40^CrePten^f T cells.

Figure 5. OX40^CrePten^f Th cells can undergo apoptosis in vitro and contract in vivo normally.

A) OT2 Pten^f or OT2 OX40^CrePten^f CD4 T cells were injected into SJL (CD45.1). The number of CD45.2⁺ donor cells in the draining LNs was counted at various time points after s.c. injection with either PBS or LPS+OVA. Data show mean±SD of a minimum of four mice from three independent experiments. Student’s t test was used to calculate p values. B) Death was assayed in CD4 T cells at 24hr intervals after in vitro activation. Data represents 2 independent experiments. C) CD4 T cells were activated in vitro for three days then cultured with no IL-2, 20ng/mL IL-2, 20ng/mL IL-2 +activation-induced death stimuli (10μg anti-CD3, 1μg/mL anti-Fas) or 20ng/mL IL-2 +DNA damage (400 rads γ-irradiation). Death was assayed after 24hrs. Data show mean ± SD for n ≥ 6 for each genotype. Student’s t test was used to calculate p values.

To test how OX40^CrePten^f T cells respond to pathogens, we transferred naïve OT2 OX40^CrePten^fRag2^−/− or OT2 Rag2^−/− control T cells into WT hosts and infected these with attenuated Lm expressing OVA (ActA-Lm-OVA). The numbers of adoptively transferred OT2 OX40^CrePten^fRag2^−/− cells appeared to be enhanced compared with controls in response to infection, though this difference was not statistically significant (Fig. 6A). However, twice as many of these cells produced IL-2 and IFNγ compared to controls three days after infection (Fig. 6B,C). We also measured the magnitude of the endogenous CD8 T cell response to ActA-Lm-OVA using MHC tetramers. More OVA-specific CD8 T cells expanded in infected mice that had received OT2 OX40^CrePten^fRag2^−/− compared to those receiving OT2 Rag2^−/− cells (Fig. 6D). In fact, WT OT2 Rag2^−/− cells failed to increase the proportion of Lm-responsive CD8 T cells, consistent with published results (52).

Figure 6. OX40^CrePten^f Th cells can enhance bacterial and tumor responses.

A-C) Mice injected with cells from OT2 Rag^−/− (n=5) or OT2-OX40^CrePten^fRag2^−/− (n=5) mice were immunized with 10⁷ CFU ActA-Lm-Ova. After 3 days, spleens were analyzed for A) the number of transferred cells and the proportion that produced B,C) IL-2 or IFNγ. D) Mice injected with PBS (n=6), OT2 Rag2^−/− (n=6) or OT2 OX40^CrePten^fRag2^−/− (n=6) were immunized with 10⁷ CFU ActA-Lm-Ova. The percent of endogenous OVA-tetramer positive CD8 T cells from blood taken on day 8 after infection is shown. Repeated measures ANOVA was used to calculate p values. E) Mice injected i.v. with OT2 Pten^fRag2^−/− (n=10), OT2 OX40^CrePten^fRag2^−/− (n=10) or PBS (n=8) were inoculated s.c. with EG7 cells. Excised tumors were weighed on day 14. Data show median values. p values were calculated using Mann-Whitney tests. Data represent two independent experiments.

These results raised the possibility that OX40^CrePten^f CD4 T cells could promote cytotoxic immune responses in vivo under conditions where WT CD4 T cells fail to make a difference. To test this possibility further, mice were inoculated with an OVA-expressing thymoma cell line (EG7) and the ability of transferred OT2 cells to limit the growth of the tumors was determined. Indeed, mice with adoptively transferred OT2 OX40^CrePten^fRag2^−/− but not OT2 Pten^fRag2^−/− T cells rejected the tumors (Fig. 6E,F). We conclude that OX40^CrePten^f CD4 T cells can promote cytotoxic immune responses potentially by activating CD8 T cells under conditions where WT CD4 T cells fail to do so.

Discussion

We have shown here that Pten is not an essential tumor suppressor in peripheral CD4 Th cells. Instead, Pten plays an important role in regulating lymphocyte homeostasis. When Pten was lost after activation, CD4 Th cells hyper-proliferated and produced higher concentrations of cytokines. We postulate that cytokine over-production turns Pten-deficient Th cells into ‘super-helpers’ that enhance inflammatory, anti-bacterial and anti-tumor responses. However, the enhanced responses could still be resolved and spontaneous disease did not develop, in contrast to pathology in many other mouse KO models of negative regulatory proteins such as CTLA-4 and Cbl (53). In addition, bone marrow chimera and Transwell studies suggest that a soluble factor led to a cell non-CD4 T cell autonomous expansion of lymphocytes in LNs. Recently it was shown that cytokines permeate the LN and induce signaling in distant bystander lymphocytes (54). Hence, excess cytokine produced by Pten-deficient Th cells might affect other naïve lymphocytes, and significantly alter lymph node homeostasis.

We confirmed in vitro and in a CHS model that Pten acts at least in part by antagonizing signaling by the PI3K p110δ. However, while p110δ inhibitors completely block activation in WT cells, they were only partially effective in Pten-deficient cells. This suggests that other isoforms can participate in a sub-dominant manner after T cell activation, but secondary to p110δ. When negative regulatory pathways are impaired, the effects of other PI3K isoforms can accrue and become more readily detectable. Possible candidates are p110α, which we have recently shown contributes to tonic Ag receptor signaling in B cells (55), or chemokine-dependent p110γ activity (19, 20, 56, 57).

Previous studies have shown that although lymphomas are found in the lymph nodes and spleens of Pten-deficient mice, they are actually derived from the thymus (48, 49). Mechanistically, this has been linked to c-myc translocation and overexpression (49, 58). Using converse experiments, we show here that Pten does not act as a tumor suppressor in mature T cells. In humans, mature T cell lymphomas are rare compared to thymic-derived lymphomas, which may reflect increased genome stability in more mature T cells. Indeed, DNA damage checkpoint regulators were dysregulated in Pten-deficient thymocytes but not peripheral T cells (48). Alternatively, there may be additional backup mechanisms to prevent lymphomagenesis in peripheral T cells. Consistent with this, leukemic oncogenes only caused transformation when ectopically expressed in hematopietic stem cells and not mature T cells (59).

Pten has previously been shown to deter autoimmunity because when it was deleted from thymocytes, autoantibodies, autoreactive T cells and lymphoid interstitial pneumonia developed (30). Autoantibodies and tissue infilitration still arose when young mice were thymectomized, which the authors suggested was caused by defective activation-induced cell death in peripheral T cells (49). However, autoreactivity in those studies could still have been caused by failed thymocyte negative selection in young animals (30, 32). We detected no evidence of spontaneous autoantibody production, autoimmunity or inflammation in our model, possibly because there were no defects in Treg suppression or because apoptosis was only mildly affected. We thus conclude Pten is not an essential repressor of apoptosis or autoimmunity stemming from mature T cells.

Although spontaneous immunopathologies did not develop in OX40^CrePten^f mice, heightened immune responses did. We previously showed that T-dependent humoral immune responses were enhanced in OX40^CrePten^f mice due to increased number and cytokine production of T_FH cells (24). Here we show that OX40^CrePten^f Th cells enhanced cellular immune responses to Lm and prevent tumour growth. Since Pten deficiency revealed a previously unrecognized potential for Th in promoting primary CD8 T cell responses (52), we propose that OX40^CrePten^f T cells are ‘super-helpers’. The concept of super-helpers has potential clinical implications because adoptive immunotherapy and prophylactic vaccination is currently aimed at modulating responding CTLs, although interest is growing in exploiting CD4 T cells as well (4). Pten-deficient super-helper Th could potentially improve clinical outcomes. Ag-specific CD4 cells have previously been shown to promote partial or complete regression in lymphopenic environments or when high numbers were infused at regular intervals alongside Ag-specific CTLs (60-62). Here we demonstrate that similar results can be achieved using a single injection of Pten-deficient Ag-specific CD4 T cells in normal hosts. Reduction of Pten activity in mature T cells using genetic or chemical means may therefore be both safe and desirable in some therapeutic settings such as tumor immunotherapy.

Abbreviations

CHS

contact hypersensitivity

Treg

Regulatory T cell

IC

IC87114

TNCB

trinitrochlorobenzene

Lm

Listeria monocytogenes

LY

LY294002