LYSOPHOSPHATIDIC ACID INHIBITS CD8 T CELL ACTIVATION AND CONTROL OF TUMOR PROGRESSION

Abstract

CD8 T lymphocytes are able to eliminate nascent tumor cells through a process referred to as immune surveillance. However, multiple inhibitory mechanisms within the tumor microenvironment have been described that impede tumor rejection by CD8 T cells, including increased signaling by inhibitory receptors. Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that has been shown repeatedly to promote diverse cellular processes benefiting tumorigenesis. Accordingly, the increased expression of LPA and LPA receptors is a common feature of diverse tumor cell lineages and can result in elevated systemic LPA levels. LPA is recognized by at least 6 distinct G-protein-coupled receptors and several of which are expressed by T cells, although the precise role of LPA signaling in CD8 T cell activation and function has not been defined. Here, we demonstrate that LPA signaling via the LPA₅ receptor expressed by CD8 T cells suppresses antigen receptor signaling, cell activation and proliferation in vitro and in vivo. Importantly, in a mouse melanoma model tumor-specific CD8 T cells that are LPA₅-deficient are able to control tumor growth significantly better than wild-type tumor-specific CD8 T cells. Together, these data suggest that the production of LPA by tumors serves not only in an autocrine manner to promote tumorigenesis but also as a mechanism to suppress adaptive immunity and highlights a potential novel target for cancer treatment.

Introduction

The adaptive immune system is able to detect and eliminate nascent tumors through a process referred to as immune surveillance and mediated in large part by cytotoxic CD8 T cells. However, this immune response to tumor may also contribute to tumorigenesis by providing selective pressure to which tumors adapt and eventually evade eradication, a process coined cancer immunoediting (1, 2). Tumors that evade the initial T cell response could create an immunosuppressive microenvironment from which variants that are able to escape immune control and grow without restraint may arise. The mechanisms used by tumors to either evade the initial CD8 T cell response or promote the tumor immunosuppressive environment are not fully defined. However, the recent success of immunotherapies that interfere with tumor-derived immune suppression (3–5) has underscored the importance of identifying the mechanisms by which tumors suppress CD8 T cells to escape immune control (6, 7).

The activation of cytotoxic CD8 T cells by either foreign or tumor antigen is initiated via signals transmitted by the T cell antigen receptor (TCR) (8). TCR signaling and the subsequent function of mature T cells can be regulated in a positive or negative manner by different surface co-receptors (9). Multiple inhibitory mechanisms within the tumor microenvironment that impede tumor rejection by tumor-infiltrating lymphocytes (TIL) have been described (6, 10), including the increased signaling by CD8 T cell inhibitory receptors, such as the well-characterized CTLA-4 molecule (11).

Lysophospholipids comprise a small family of structurally simple lipids that include sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), and they induce diverse biological and pathophysiological effects by signaling through specific G-protein-coupled receptors (GPCR) (12, 13). The LPA lysophospholipid is recognized by 6 specific GPCR, LPA_1–6 (14), and although T lymphocytes are known to express several LPA receptors (13, 15–17), the immune regulatory activities of LPA are not well-understood. LPA concentration in the blood of healthy individuals has been reported to vary from high nanomolar to low micromolar levels (12, 18). In vivo production of LPA results predominantly from the activity of autotaxin (ATX) (19), an extracellular lysophospholipase D originally isolated and identified from a human melanoma as an autocrine motility factor (20). Since then LPA has been found aberrantly produced in a number of different malignant cell types (21–23) resulting in significantly increased systemic levels that can reach 60 µM in malignant effusions (24–26). At these elevated levels LPA has been shown to promote tumor progression by enhancing tumor migration, survival, metastasis, angiogenesis, and therapeutic resistance (27–31).

Previously LPA has been shown to modulate the activation of different cell types (17), and in this study we investigated if LPA could influence CD8 T cell activation. Here we report that CD8 T cells express the LPA₅ receptor and signaling by this GPCR inhibits CD8 T cell receptor signaling, activation and proliferation. Furthermore, we demonstrate that tumor-specific CD8 T cells lacking LPA₅ can control the progression of established tumor more efficiently than the LPA₅-sufficient tumor-specific CD8 T cells. Thus, our findings reveal a novel role for lysophospholipid-mediated protection of tumor from adaptive immunity.

Materials and Methods

Mice

C57BL/6 (CD45.2) and CD45.1 (B6.SJL-Ptprc^a Pepc^b/BoyJ) were obtained from Jackson Laboratories or bred in the Biological Resource Center (BRC) at National Jewish Health (NJH). CD45.1 OT-I mice (gift of Dr. Ross Kedl, University of Colorado), Lpar2^−/− mice (gift of Dr. Jerold Chun, Scripps Research Institute), Lpar5^−/− mice, TCRα^−/− mice (gift of Dr. Philippa Marrack, NJH) were bred in the BRC at NJH. LPA₅^−/− mice were generated as described in Supplementary Data. All mice used were 8–12 weeks of age, housed under specific pathogen-free conditions and were maintained in accordance with the regulations of the Institutional Animal Care and Use Committee.

Calcium mobilization

Erythrocyte-lysed spleen, pooled lymph nodes, or purified CD8⁺ T cells were suspended at 20 × 10⁶ cells/mL in RPMI 1640 medium supplemented with 2.5% fatty-acid free (faf) BSA (Calbiochem) and loaded with Indo1-AM (Molecular Probes) as described in Supplementary Data.

qPCR

CD8⁺ T cells were isolated from the spleens and lymph nodes of wild-type mice and LPA receptor expression measured by quantitative real-time RT-PCR using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen Life Technologies). The details of qPCR analysis including specific forward and reverse LPA receptor primers are provided in the Supplementary Data.

Flow cytometry

All antibodies were purchased from eBiosciences, BD Pharmingen, Biolegend, or were produced in our laboratories. Cells were stained in 2% BSA-PBS+0.1% sodium azide with blocking Fc receptor antibody (2.4G2) on ice for 20–30 minutes. For viability assessment, 7-AAD was added 10 minutes prior to data acquisition. All flow cytometric analysis was performed on the LSRII flow cytometer (BD) and analyzed with FlowJo v8 (Tree Star) and GraphPad Prism software (v 5.0).

Lipid preparation

LPA (16:0 AGP, Avanti Polar Lipids) was solubilized to 5 mM concentration in 0.1% BSA-PBS, aliquoted, and stored at −20C. Aliquots were diluted to 1mM in RPMI 1640 medium supplemented with 2.5% faf-BSA (Calbiochem) prior to use. OTP was generated as previously described (32), stored as a powder, and solubilized in 95% methanol to create aliquots. Virgin glass tubes and caps were sterilized by autoclave for aliquots that were stored at −20C. The concentration was confirmed by phosphorus assay. OTP was solubilized for experimental use by sonication with FBS- or BSA-containing culture media or vehicle (2% propanediol, 1% ethanol in PBS) for in vitro or in vivo usage, respectively. For in vitro experiments, OTP was solubilized to 50 µM and passed through a 0.2 µm filter for further sterilization. For in vivo experimentation, solubilized OTP was transferred to siliconized eppendorf tubes and animals were dosed at 5 mg/kg every 8 hours.

Generation of bone marrow-derived dendritic cells

Congenic gender-matched bone marrow-derived dendritic cells (BMDC) were generated by flushing of femur and tibia and culture at 10⁶ cells/mL in RPMI 1640 with 20 ng/mL GM-CSF, 10% FBS (Omega Scientific), Penicillin-Streptomycin, and GlutaMAX (Invitrogen). Media was refreshed on days 3 and 5. On day 7, BMDC were harvested from culture and stimulated with 1 ng/mL LPS for 90 minutes and pulsed with peptide for the last hour of LPS treatment. BMDC were washed 5 times to remove LPS and unbound peptide before transfer.

In vitro T cell activation and proliferation

To determine how LPA affected antigen-specific activation of CD8 T cells, OT-I splenocytes were isolated, erythrocyte lysed, and labeled with CFSE (Invitrogen). For all CFSE labeling, cells were suspended at 15 × 10⁶ cells/mL in PBS and CFSE was added to a final concentration of 2 µM for 10 minutes and then washed in media. Splenocytes were pulsed with 1 µM of the SIIGFEKL (G4, Anaspec, Inc.) or SIINFEKL (gift of Philippa Marrack) peptides for 4 hours or 90 minutes, respectively, in 5% faf-BSA RPMI, then washed. Cells were cultured in 96 well plates at 2.5 × 10⁶ cells/mL in the presence or absence of 50 µM OTP that was sterile-filtered prior to addition to culture. Cells were enumerated by flow cytometry and the proportion of cells proliferated was calculated by Flowjo analysis. The MFI values of activation marker expression were normalized.

To assess in vitro cytokine production, OT-I effector T cells were generated by pulsing erythrocyte-lysed OT-I splenocytes with 1 µM SIINFEKL and culture with IL-2 for 5 days. On day 5 of culture, target cells (EL4 cells) were pulsed with 1 µM SIINFEKL and cultured at an effector to target ratio of 0.625:1 with OT-I effector T cells for 4 hours in the presence of Brefeldin A, in the presence or absence of sterile-filtered 50 µM OTP.

In vivo T cell transfer and antigen-specific stimulation

BMDC were generated as described above. One day prior to BMDC transfer, CD8⁺ T cells were purified from OT-I spleen and LN cells with a CD8⁺ enrichment kit (Miltenyi) to a purity of ≥95%, and 10⁶ CFSE-labeled CD8⁺ T cells were transferred to CD45 allotype-mismatched recipient C57BL/6 mice. SIINFEKL-BMDC (10⁶) were suspended in PBS and transferred s.c. in the scruff to individual recipients. On d3 post-immunization, animals were sacrificed and dLN (axilary, brachial, cervical), ndLN (inguinal, mesenteric), and spleen were harvested. After erythrocyte lysis, cells were counted by Z2 Coulter Particle Count and Size Analyzer (Beckman-Coulter) and 10 × 10⁶ cells were stained for flow cytometry. Cells were suspended in FACS buffer and stained with 7-AAD for viability before analysis on the BD LSRII.

B16.cOVA tumor experiments

The OVA-transfected B16 tumor cell line (B16.cOVA) was kindly provided by Dr. Ross Kedl. Cells were maintained in RPMI 1640 (Cellgro), supplemented with 10% heat-inactivated FBS (Omega Scientific), GlutaMAX, Penicillin-Streptomycin, MEM NEAA, sodium pyruvate (Invitrogen), and 0.75 mg/mL G418 sulfate selection (Cellgro).

To determine how LPA₅^−/− OT-I T cells responded to tumor, 10⁵ B16.cOVA cells were transferred s.c. into the hind leg of recipients and 5 days later 0.5 × 10⁶ CFSE-labeled OT-I T cells were transferred s.c. into the tail vein. Recipients were sacrificed 5 days later and tumor and dLN were harvested. Lymphoid organs were mashed through a cell strainer (100 µm, BD), erythrocyte lysed, and counted. Tumors were digested in 0.5 mg/mL Collagenase D (Fisher Scientific) and 60 U/mL DNase (Sigma-Aldrich) for 30 minutes with perturbation every 10 minutes, and then neutralized with 5 mM EDTA for 5 minutes. Tumor cells were homogenized by pipette tituration and passed through a 100 µm filter, erythrocyte lysed, and counted. Tumor diameter was measured with calipers and body weight and overall appearance were assessed every 2 days. Mice were sacrificed when tumor diameter exceeded 10 mm or at 8 days after T cell transfer, whichever came first.

Statistical analysis

All statistical analysis was performed with GraphPad Prism software (version 5.0) using a two-tailed unpaired Student’s t-test.

Results

LPA inhibits T cell antigen receptor-mediated calcium mobilization via LPA₅

Both human and murine T cells express LPA receptors (15, 16) so we initially questioned if LPA could influence TCR signaling. Specifically, we tested if LPA could modulate TCR-induced intracellular calcium mobilization. These results revealed that 20 µM LPA potently inhibited intracellular calcium mobilization by CD8⁺ T cells in response to TCR stimulation achieved by antibody-mediated crosslinking of the TCR (Supplementary Fig. S1). In contrast, LPA treatment in the absence of antigen receptor stimulation did not affect intracellular calcium levels. LPA inhibition of CD8 TCR signaling was further shown to be dose-dependent with a reduction of intracellular calcium levels achieved with LPA concentrations as low as 1 µM (Fig. 1A), approximating the levels reported in the blood of healthy individuals (12, 18).

Figure 1.

LPA signals via LPA₅ inhibit CD8⁺ T cell TCR-mediated intracellular calcium release. A, Intracellular calcium concentration was assessed in purified CD8⁺ T cells stimulated with 10 µg/mL anti-CD3 in the absence (black line) or presence of the indicated titrated concentrations of LPA. B, Expression of Lpar1-6 by isolated CD8⁺ T cells as measured by qPCR. C, LPA₂^−/− or LPA₅^−/− CD8⁺ T cells were isolated and intracellular calcium mobilization was assessed in response to anti-CD3 stimulation in the presence (bold line) or absence (thin line) of 20µM LPA.

There are currently six validated LPA receptors: LPA_1–6 (14) and quantitative PCR analyses revealed that purified CD8 T cells expressed LPA₂ , LPA₅ and LPA₆ (Fig. 1B), consistent with previous findings (15, 16). To determine which LPA receptor(s) was responsible for LPA inhibition, we compared calcium mobilization in TCR-stimulated CD8⁺ T cells from LPA₂^−/− and LPA₅^−/− mice (LPA₆^−/− mice were not commercially available). These results revealed that although LPA inhibition was intact in the LPA₂-deficient T cells, LPA inhibition was absent in LPA₅-deficient T cells (Fig. 1C). These data demonstrate that LPA₅ expression is required for the inhibition of TCR-mediated calcium mobilization.

LPA signaling inhibits proliferation and activation in vitro

Increased intracellular calcium concentration is an important early consequence of antigen receptor signaling that leads to the activation of distinct transcriptional programs important for T cell activation and function (33). Thus, we next tested whether LPA suppression of TCR-induced calcium mobilization would lead to reduced CD8 T cell activation in vitro. Initial experiments revealed that LPA was able to inhibit TCR-mediated activation of CD8⁺ T cells, as determined by reduced CD62L down regulation(34), however only when T cells were stimulated with sub-optimal concentration of anti-CD3 (Supplementary Fig. S2). To investigate if LPA could inhibit antigen-specific stimulation of CD8 T cells, we used the well-characterized OT-I TCR transgenic mouse model that features CD8 T cells that express a TCR specific for the chicken ovalbumin peptide, SIINFEKL, in the context of the MHC molecule, H-2K^b (35). This system also allowed us to modulate T cell activation with different stimulatory peptides (36). More specifically, the OT-I TCR displays relatively high affinity (K_D ∼5 µM) for the SIINFEKL ovalbumin peptide but it also recognizes the altered peptide ligand, SIIGFEKL (G4), with relatively weaker affinity (K_D ∼10 µM) (37). Thus with these peptides we could compare the effects of LPA signaling after relatively high or low affinity TCR stimulation. Because LPA is degraded both in vitro and in vivo (38), we also used a metabolically stabilized LPA analog, octodecenyl thiophosphate (OTP) to induce LPA signaling (39, 40). Of note, OTP is preferentially recognized by LPA₅ relative to other LPA receptors and displays a much lower EC₅₀ for LPA₅ compared to other LPA receptors (41).

Using this system we monitored CD8 T cell proliferation and expression of activation markers after antigen-specific TCR stimulation in vitro in the absence or presence of LPA signaling achieved with OTP. Proliferation was monitored by CFSE dilution and showed that in the presence of OTP, CD8 T cells were acutely inhibited from proliferating in response to G4 peptide stimulation (Fig. 2A). Specifically, by day 3 control cells had typically started to divide with the majority having undergone at least 2 divisions by day 7 while the majority of OTP-treated cells remained undivided in the same period. OTP inhibition of CD8 T cell activation was further evidenced by the significantly reduced expression of both the CD25 and CD44 activation antigens after TCR stimulation (Fig. 2B). Likely as a consequence of inhibiting activation and proliferation, OTP treatment also resulted in reduced accumulation of CD8⁺ T cells (Fig. 2C) without any significant effect on viability as compared to unstimulated controls (Fig. 2D). Importantly, OTP treatment did not affect the ability of antigen presenting cells to present peptide to OT-I T cells as OTP-treated and -untreated antigen presenting cells were equally able to induce CD25 upregulation (Supplementary Fig. S3). Similar to our initial experiment with anti-CD3 stimulation, we found that the ability of LPA signaling to dampen CD8 T cell proliferation and activation was less effective after the TCR was stimulated with the higher affinity SIINFEKL peptide (Fig. 2E). In addition, OTP treatment did not appear to significantly influence SIINFEKL-stimulated TNFα or IFNγ cytokine production by in vitro-generated effector CD8⁺ T cells (Fig. 2F). Together these findings show that LPA signaling potently suppresses TCR signaling, cell activation and proliferation when T cells are stimulated by relatively weak affinity antigens.

Figure 2.

LPA signaling inhibits in vitro TCR-induced activation and proliferation. A, CD8⁺ T cells were stimulated with specific antigen in the absence (upper row) or presence (middle row) of OTP and analyzed for the expression of the CD25 activation antigen and CFSE fluorescence at the indicated times. Overlay of CFSE histograms by day are shown in the bottom row in the absence (thin line) or presence of OTP (bold line) or when left unstimulated (gray filled). Data shown are representative of 3 independent experiments. B, CD25 and CD44 expression on CD8⁺ T cells is shown in histograms after 3 or 4 days, respectively, of in vitro culture in the absence of stimulation (gray filled) or after G4 peptide stimulation alone (thin line), or in the presence of OTP (bold line). Histograms are representative of 3 independent experiments and normalized MFI values are summarized in bar chart (Mean + SEM, **p<0.01, ***p<0.0001). C, Enumeration of in vitro cultured peptide-specific OT-I CD8⁺ T cells at the indicated times in the absence of peptide (grey circles) or after peptide stimulation alone (open circles) or in the presence of 20 µM OTP (black circles). D, Cell viability was determined by 7-AAD staining at the peak of CD25 expression and data are representative of 3 independent experiments (Mean + SEM). E, Splenocytes from OT-I transgenic mice were labeled with CFSE, stimulated with SIINFEKL peptide and cultured for 3 days before analyses of CFSE fluorescence. F, Effector OT-I CD8⁺ T cells were unstimulated (left) or re-stimulated with SIINFEKL in the absence (center) or presence (right) of OTP before analysis of TNFα and IFNγ expression. Data shown are representative of 2 independent experiments.

Increased LPA signaling dampens activation and proliferation in vivo

Our in vitro data demonstrate that LPA signaling via LPA₅ on CD8 T cells inhibits TCR signaling, activation, and proliferation. We next addressed whether this LPA regulatory pathway operated similarly in vivo. To accomplish this, we transferred purified, CFSE-labeled OT-I CD8⁺ T cells into C57BL/6 recipients followed by antigen-specific stimulation achieved by the subsequent transfer of bone-marrow-derived dendritic cells (BMDC) previously pulsed with TCR-specific peptide (Fig. 3A). Under these conditions, BMDC pulsed with the relatively weak affinity G4 peptide were unable to stimulate OT-I CD8⁺ T cells as determined by the absence of increased activation antigen expression or proliferation (data not shown). In contrast, when SIINFEKL peptide-pulsed BMDC (SIINFEKL-BMDC) were transferred, OT-I CD8 T cell proliferation was observed clearly 3 days later in the draining lymph nodes (dLN; axillary, brachial, cervical) relative to the site of BMDC transfer (Fig. 3B).

Figure 3.

LPA signaling inhibits TCR-mediated CD8⁺ T cell activation in vivo. A, Schematic of adoptive transfer of OT-I CD8⁺ T cells and in vivo OTP treatment. B, Histograms of CFSE dilution in vehicle-treated control (thin line), OTP-treated (bold line), and unstimulated control (gray filled) OT-I CD8⁺ T cells isolated from dLN. C, Number of OT-I CD8⁺ T cells in dLN. D, Representative histograms of CD25 expression by OT-I CD8⁺ T cells after vehicle-treatment (thin line), OTP-treatment (bold line) or unstimulated (grey shaded). Right panel shows expression of CD25 as mean fluorescence intensity (MFI) by OT-I CD8⁺ T cells in the dLN from individual mice after vehicle-treatment (closed circles) or OTP-treatment (open circles). Data in B-D are representative of 2 independent experiments (n=4 mice/group, mean + SEM, **p<0.01).

To promote LPA signaling in vivo we treated mice with OTP subcutaneously, which results in detectable levels in the blood after one hour with an approximate half-life of 5.5 hours (GT, unpublished data). Mice were treated with 5 mg/kg OTP every 8 hours, with the first dose preceding SIINFEKL-BMDC transfer by one hour (Fig. 3A). Three days after antigen-specific stimulation, the OT-I CD8 T cells recovered from the dLN of OTP-treated animals had proliferated considerably less compared with those from the vehicle-treated animals (Fig. 3B). Furthermore, the number of OT-I CD8⁺ T cells recovered in the dLN of OTP-treated mice was significantly reduced relative to those recovered from the vehicle-treated animals and consistent with an inhibition of proliferation (Fig. 3C). Additionally, antigen-specific T cell activation was diminished as evidenced by the reduced expression of CD25 by the transferred OT-I CD8 T cells in OTP-treated mice compared to those of the vehicle-treated mice (Fig. 3D). Taken together, these findings demonstrate that increased LPA signaling inhibits antigen-specific CD8 T cell proliferation and activation both in vitro and in vivo.

LPA₅-deficient CD8 T cells show enhanced proliferation in response to antigen-specific stimulation in vivo

To directly test if LPA₅ signaling suppressed CD8 T cells in vivo, we measured antigen-driven proliferation of LPA₅-deficient and wild-type OT-I CD8 T cells after adoptive transfer. LPA₅^−/− mice are phenotypically unremarkable compared to wild-type littermates and display comparable numbers and frequencies of splenic CD8⁺ and CD4⁺ T cell populations (Supplementary Fig. S4), similar to that reported for an independently generated LPA₅-deficient mouse strain (42). Furthermore, in vitro LPA₅-deficient CD8 T cells displayed similar viability as wild type cells (data not shown). To address whether LPA inhibited antigen-specific CD8 T cell responses under normal physiological conditions, LPA₅^+/+ or LPA₅^−/− OT-I CD8⁺ T cells were isolated, CFSE-loaded, transferred into recipients, which were subsequently immunized with SIINFEKL-pulsed BMDC similar to those described in Fig. 3A except neither group was treated with OTP. The results from these experiments show that 3 days after antigen-specific stimulation, a considerable proportion of wild type OT-I CD8 T cells had undergone cell division. In contrast, similar stimulation of LPA₅-deficient OT-I CD8 T cells resulted in an increased percentage of LPA₅-deficient OT-I CD8 T cells that had undergone cell division and fewer transferred cells that remained CFSE^high (Fig. 4A). Consistent with an increased frequency of LPA₅^−/− OT-I CD8 T cells having proliferated, higher numbers of mutant OT-I CD8 T cells were recovered from the dLN compared to wild-type cells (Fig. 4B). Together these data reveal that in the absence of LPA₅-mediated suppression, antigen-specific stimulation of OT-I CD8 T cells leads to a higher frequency of cells that proliferate and accumulate in the dLN.

Figure 4.

LPA₅-deficient CD8⁺ T cells stimulated by peptide in vivo exhibit enhanced proliferation and accumulation. A-B, Purified LPA₅^+/+ or LPA₅^−/− OT-I CD8⁺ T cells were CFSE labeled and transferred into C57BL/6 recipients, similar to that described in Fig. 3A. Recipients were immunized one day later with SIINFEKL-pulsed BMDCs. On day 3 post-immunization, mice were sacrificed and draining lymph nodes harvested for analysis. A, Proliferation of LPA₅^+/+ (thin line), LPA₅^−/− (bold line) or unimmunized (filled gray) OT-I CD8⁺ T cells as indicated by CFSE dilution. Data shown are representative of 2 independent experiments. B, Number of OT-I CD8⁺ T cells harvested from the dLN of LPA₅^+/+ (closed circles; n=8) and LPA₅^−/− (open circles; n=8) OT-I CD8⁺ T cell recipients in the absence of stimulation or 3 days after peptide stimulation (p=0.053). Data shown are cumulative of 2 independent experiments.

Transfer of LPA₅-deficient tumor-specific CD8 T cells controls tumor progression

Our data thus far show that LPA signaling via LPA₅ expressed by CD8 T cells suppresses T cell activation and proliferation. A number of different tumor cell types have been reported to produce elevated levels of LPA that promote survival, growth and tumorigenesis (26, 43). Thus, we next compared the ability of LPA₅-deficient and LPA₅-sufficient tumor-specific CD8 T cells to control progression of an established tumor.

To address this question, we used the B16 melanoma cell line, B16.cOVA, which stably expresses chicken ovalbumin (with the SIINFEKL peptide) and used OT-I T cells as tumor-specific CD8 T cells as previously reported (44). B16.cOVA cells were subcutaneously implanted in the rear leg of wild-type C57BL/6 mice and 5 days later either naïve LPA₅^−/− or wild type OT-I CD8⁺ T cells were transferred into these recipients and the tumor diameter measured every 2 days thereafter (Fig. 5A). The results from these experiments showed that tumors grew similarly in the presence or absence of wild-type OT-I CD8 T cell transfer (Fig. 5B and 5E), consistent with previous reports demonstrating tumor progression in the presence of wild-type tumor-specific CD8 T cells (45, 46). In contrast, tumor growth was clearly abated in mice 6 and 8 days after receiving LPA₅-deficient OT-I CD8⁺ T cells (Fig. 5B and 5E). Compared to the wild type tumor-specific CD8 T cells, LPA₅-deficient tumor-specific CD8 T cells were also found typically at higher numbers within the tumor (Fig. 5C). However, compared to the wild type CD8 T cells, these LPA₅-deficient tumor-infiltrating CD8 T cells appeared to express similar levels of IFNγ, TNFα, the inhibitory PD-1 receptor and the CCR7 chemokine receptor 5 days after transfer (Fig. 5D and data not shown). Averaging tumor size across a treatment group demonstrated that while the tumors were similar in size at the time of T cell transfer, a significant reduction in tumor size was observed at 6 and 8 days post-transfer of LPA₅^−/− tumor-specific CD8 T cells compared to the tumor size in mice transferred with wild-type CD8 T cells (Fig. 5B). Further evidence that tumor-specific LPA₅^−/− CD8 T cells were better able to control tumor progression compared to wild-type CD8 T cells was provided by the significantly smaller tumors that were harvested 8 days post T cell transfer (Fig. 5E and 5F). These data demonstrate that tumor growth is inhibited to a greater degree when LPA₅ signaling is prevented in tumor-specific CD8 T cells.

Figure 5.

Enhanced control of tumor progression by LPA₅-deficient CD8⁺ T cells. A, Schematic of tumor establishment and subsequent OT-I CD8⁺ T cell adoptive transfer. C57BL/6 recipients were implanted s.c. in the leg with 10⁵ B16.cOVA cells. Five days post-implantation, 0.5 × 10⁶ LPA₅^+/+ or LPA₅^−/− OT-I CD8⁺ T cells were transferred i.v. and tumor progression evaluated at the specified timepoints. B, Average combined tumor burden at the indicated days in the absence of T cell transfer (gray circles; n=9) or after adoptive transfer of LPA₅^+/+ OT-I (black, n=10), LPA₅^−/− OT-I (white, n=11) or untreated controls (gray, n=9). Data are cumulative of 3 independent experiments (mean +/− SEM, *p<0.05, **p<0.01). C, Number of tumor-specific CD8⁺ T cells per gram of tumor at day 5 after adoptive transfer of LPA₅^+/+ (black, n=11) or LPA₅^−/− (white, n=11) OT-I CD8⁺ T cells. Data are cumulative of 3 independent experiments (mean +/− SEM, p=.08). D, Tumors were harvested on day 5 after T cell transfer and LPA₅^+/+ (black line) and LPA₅^−/− (dotted line) OT-I CD8 T cells evaluated for IFNg and TNFa expression and compared to tumor-free wild type lymph node OT-I T cells (grey histogram). E, Tumor mass 8 days after adoptive transfer of LPA₅^+/+ OT-I (black, n=10), LPA₅^−/− OT-I (white, n=11) or untreated controls (gray, n=9). Data are cumulative of 3 independent experiments (mean +/− SEM, **p<0.01). F, Representative tumors in situ and ex vivo from C57BL/6 mice in the absence of T cell transfer or 8 days after adoptive transfer of LPA₅^+/+ or LPA₅^−/− tumor-specific CD8⁺ T cells.

Discussion

CD8 T cells are specialized cells of the adaptive immune system with the ability to recognize and eliminate nascent tumors. However, tumors are notorious for promoting an immunosuppressive environment through different mechanisms that thwart this adaptive immune response. Notably, the identification and targeting of some of these immunosuppressive mechanisms has led to relative success in the immunotherapeutic treatment of melanoma (3–5). Our data reveal a previously uncharacterized ability of the LPA lysophospholipid to suppress CD8 TCR signaling and in vivo activation, proliferation and tumor control. As aberrant production of LPA is a common feature of diverse cancer cell types, these data further suggest that tumor cells can exploit a naturally-occurring lipid to not only promote tumorigenesis but also to create an immunosuppressive environment.

Precedence for lysophospholipid regulation of adaptive immunity has been established previously in studies of S1P, which plays an important role in directing lymphocyte trafficking, localization and development (47, 48). Similar to S1P, LPA is recognized by multiple GPCR that are differentially expressed by lymphocytes; in previous reports using primary human CD4 T cells and T cell lines, LPA have been shown to regulate intracellular calcium mobilization (49) and cytokine production (16, 50). However, these in vitro studies did not identify the precise T cell signaling pathway regulated by LPA, or which LPA receptor mediated these activities. In this report we demonstrate that LPA engagement of LPA₅ suppresses CD8⁺ T cell receptor signaling, activation and tumor immunity.

CD8 T cells express several LPA receptors, and we have identified LPA₅ expression to be required for not only negative regulation of TCR-induced calcium mobilization but also for inhibiting in vivo antigen-mediated proliferation. TCR-mediated increase in cytosolic calcium is an early signaling event important for both proximal and distal CD8 T cell activities (33). Indeed, the primary encounter between antigen-specific CD8 T cells and the specific antigen is known to program the subsequent proliferation and differentiation of antigen-specific cells in vitro and in vivo (51, 52). Analyses of TCR-mediated intracellular calcium mobilization unambiguously demonstrated the ability of LPA to suppress TCR signaling at 1 µM, the lowest concentration tested, which approximates the physiological level of LPA in normal individuals (18). At higher concentrations of LPA (or OTP), antigen-mediated CD8 T cell activation and proliferation in vitro were considerably suppressed. However, it is not yet clear if the source of LPA that suppresses CD8 T cell tumor immunity is derived from the tumor, the endogenous systemic levels, or from elsewhere, which is currently under investigation in our lab. It is important to note that the significance of the systemic levels of LPA in regard to LPA receptor-signaling is not yet established. Current models suggest that biologically relevant LPA signaling results from localized and directed autocrine/paracrine LPA production and signaling (19, 53), where concentrations can reach considerably higher levels. In this model, secreted autotaxin has been proposed to associate with integrins on a cell surface where LPA is locally produced and engages nearby LPA receptors.

To assess the ability of LPA – LPA₅ signaling to suppress antigen-specific CD8 T cell responses in vitro and in vivo, we used an established model system that allowed us to either increase LPA signaling using an LPA analog, OTP, or prevent LPA inhibitory signaling using LPA₅-deficient CD8 T cells. Our in vitro data suggest that LPA suppression of antigen-specific CD8 T cells operates more efficiently for relatively weak affinity peptide antigens (Fig. 2 and Suppl. Fig. 2). However, in our studies neither wild type nor LPA₅^−/− OT-I CD8 T cells were stimulated in vivo with BMDC pulsed with the lower affinity G4 peptide. Thus, whether LPA₅ suppression operates particularly for relatively weak affinity antigens could not be confirmed in vivo but this is an area of ongoing investigation. As endogenous tumor-specific CD8 T cells normally express TCR with relatively low affinity for tumor antigens (54–56), this would suggest that tumor-derived LPA may be particularly effective at suppressing CD8 T cell tumor immunity.

Our results clearly demonstrate that LPA signaling via LPA₅ inhibits CD8 T cell TCR signaling, activation and proliferation, and that LPA₅-deficient tumor-specific CD8 T cells are better than the wild type cells at controlling the growth of established tumor . However, analysis of tumor infiltrating CD8 T cells 5 days after adoptive transfer did not reveal any apparent difference between tumor-specific LPA₅^−/− and wild type CD8 T cells with respect to the expression of IFNγ, TNFα, PD-1 inhibitory receptor, or the CCR7 chemokine receptor. Nevertheless, when stimulated with specific antigen in vivo, LPA₅-deficient CD8 T cells consistently proliferated to a greater extent and accumulated to higher numbers relative to wild type CD8 T cells (Fig. 4 and Fig. 5C). Thus, together these data support a model in which LPA₅^−/− tumor-specific CD8 T cells are more easily stimulated to proliferate in response to tumor antigen and the increased proliferation and accumulation equate, at least in part, to better tumor control. We note, however, that LPA has been shown to influence T cell homing (57) and migration (58). Thus, it remains possible that LPA₅^−/− tumor-specific CD8 T cells also display altered migration and trafficking, or they could influence CD8 T cell function by other indirect mechanism(s) and which is the focus of ongoing experiments.

As enhanced LPA production is a feature of many malignant cell types, our findings further suggest that in addition to the role of this lipid in enhancing tumorigenesis (27–29), LPA production by these cells also represents an additional inhibitory mechanism within the tumor microenvironment that serves to impede tumor rejection by CD8 T cells. The recent success of immunotherapies that antagonize inhibitory receptor signaling by T cells suggests that identification and characterization of additional signaling pathways used by tumors to suppress CD8 T cell tumor immunity will only improve cancer immunotherapy (6, 7). Data presented here demonstrate that LPA₅ functions as an additional inhibitory receptor on CD8 T cells. Given the association of LPA with multiple cancers (26, 43, 59), blockade of LPA₅ signaling may be a promising additional strategy to promote host CD8 T cell tumor immunity.