SEARCH

SEARCH BY CITATION

Keywords:

  • Cancer;
  • Cell death;
  • Dendritic cells;
  • Regulatory T cells;
  • Tumor immunology

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Reagents
  8. Acknowledgements
  9. Conflict of interest
  10. References
  11. Supporting Information

Intrinsic immunosuppression is a major obstacle for successful cancer therapy. The mechanisms for the induction and regulation of immunosuppression in humans are ill defined. A microenvironmental component that might prevent antitumor immunity is the presence of dying tumor cells, which are abundant following conventional cancer ablation methods such as chemo- or radiotherapy. Shedding of apoptotic debris and/or secretion of factors to the tumor bed or draining lymph nodes thus might have a profound impact on professional phagocytes, such as DCs, and subsequent priming of lymphocytes. Here, we exposed human DCs to supernatants of live, apoptotic, or necrotic human breast cancer cells and cocultured them with autologous T cells. Priming with apoptotic debris prevented DCs from establishing cytotoxicity toward live human tumor cells by inducing a Treg-cell population, defined by coexpression of CD39 and CD69. Immunosuppression via Treg cells was transferable and required the release of sphingosine-1-phosphate (S1P) from apoptotic cells, acting via S1P receptor 4 on DCs to induce IL-27 secretion. We propose that CD69 expression on CD39+ Treg cells enables them to interact with CD73-expressing CD8+ T cells to generate adenosine, thereby suppressing cytotoxicity. These findings aid the understanding of how dying tumor cells limit antitumor immunity.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Reagents
  8. Acknowledgements
  9. Conflict of interest
  10. References
  11. Supporting Information

A growing tumor activates the immune system in ways to ensure its own survival and encourage the formation of metastases. Polarization to a tumor-supportive state occurs for APCs of the innate immune system, such as macrophages or DCs [[1]]. These phagocytes program adaptive immunity by generating tumor-specific Treg cells, which are a major obstacle for antitumor immunity [[2]]. Treg cells are primed and activated mainly in the tumor-adjacent draining lymph nodes (TDLNs) by factors shed from the tumor [[3]]. Once primed, Treg cells travel to the tumor site, where they prevent effector T cells from eradicating the tumor [[4]]. Thus, TDLN-derived Treg cells potentially curb the benefit of an adoptive immunotherapy regime by suppressing the function of cytotoxic T cells or preventing immune activation following conventional therapy [[5]]. Hence, mechanisms of Treg-cell generation within the TDLNs have to be defined in order to design effective therapeutic strategies.

Generation/priming of tumor-specific Treg cells, requires antigen uptake and presentation by professional APCs, that is, DCs. Tumor-derived antigens are acquired by DCs directly at the tumor site, from metastasing tumor cells [[6]], or through tumor-released exosomes/microvesicles [[7]], which are primarily drained to the TDLNs. In addition to antigens, DCs receive other tumor-derived signals that shape their phenotype and the subsequent profiling of T cells [[1, 4]]. Depending on the tumor microenvironment, DCs may exist in different states of maturation and activation [[6]].

A prominent microenvironmental niche in tumors are dying tumor cells [[8]]. Extremes of the immunological outcome of tumor-cell death are (i) antigen cross-presentation by DCs that prepares immune effectors to eradicate malfunctioning cells or (ii) tolerance to dampen an overactivated immune reaction [[9]]. The decision toward inflammation versus tolerance depends on the surface protein/lipid signatures exposed by dying cells, which are determined by the mode of cell death [[10]]. For instance, necrosis induces shedding of danger-associated molecular patterns, which activate TLRs on DCs [[10]]. The immunological outcome of apoptosis is comparatively ambiguous. Apoptosis can be immunogenic as demonstrated by an increase in antigen cross-presentation and the induction of cytotoxic T cells upon priming with ACs in vivo [[11]]. On the other hand, triggering of multiple immunosuppressive pathways upon priming with ACs has been recognized [[12]]. In case of cancer ablation treatments such as chemotherapy, the decision of generating an antitumor response or tolerance might be determined by the drug, as recent evidence suggests that certain chemotherapeutic drugs trigger immunogenic cancer cell death [[13]]. However, cross-presentation of apoptotic cell (AC) derived antigens after chemotherapy does not necessarily culminate in antitumor immunity [[14]]. Besides the surface alterations on dying cells, signaling molecules secreted from dying cells that drain the adjacent lymph nodes together with tumor antigens may also be important for inducing tolerance, and possibly favor relapse [[3]]. ACs secrete immunomodulators in a regulated manner, among them lipids such as lysophosphatidylcholine or sphingosine-1-phosphate (S1P), anti-inflammatory proteins such as transforming growth factor (TGF)-β as well as nucleotides, which have the capacity to modify DC-dependent immunity [[10]]. Understanding how priming by dying cells impacts antitumor immune responses might benefit cancer therapy.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Reagents
  8. Acknowledgements
  9. Conflict of interest
  10. References
  11. Supporting Information

Apoptotic cell priming suppresses DC-dependent tumor-cell killing

We asked how DC priming by factors/exosomes shed from dying versus living tumor cells affects their ability to initiate tumor cell-specific cytotoxic T-cell responses. Conditioned media of apoptotic, necrotic, or viable MCF-7 cells (apoptotic tumor cell condition medium, ACM; necrotic tumor cell condition medium, NCM; viable tumor cell condition medium, VCM) were incubated with monocyte-derived human DCs at a ratio of one tumor cell/DC. Higher ratios of tumor cells/DC induced cell death in DCs (Supporting Information Fig. 1A). This might be a mechanism of suppressing DC-dependent immunity when dying tumor cells are abundant, for example, after chemo/radiotherapy.

image

Figure 1. Influence of cancer cell supernatants on DC-dependent T-cell cytotoxicity. (A) Human monocyte-derived DCs were incubated with supernatants of viable (VCM), necrotic (NCM), or apoptotic (ACM) MCF-7 cells at a 1:1 ratio (supernatants of 2 × 105MCF-7/2 × 105DCs) for 16 h. Subsequently, supernatants were removed by washing, autologous T-cell-enriched PBMCs were added at a 10:1 ratio (T cells/DC) and cocultured for 3 days. The resulting T cells were then incubated with living MCF-7 tumor cells for 4 h to determine cytotoxicity. Experimental interventions as outlined in the manuscript are indicated as dotted arrows. (B) CellTracker Blue stained MCF-7 target cells were incubated with effector T cells from individual cocultures (DCs conditioned with ACM, VCM, NCM) at effector to target (E:T) ratios of 0.1:1, 0,5:1, 1:1, 1:2, 1:5, 1:10, 1:20 for 4 h. Cytotoxicity, calculated for the ACM, VCM, NCM groups, was compared with that of the control group (T cells incubated with nonconditioned DCs). Data are shown as means + SEM, representing five individual donors and experiments. (C) CellTracker Blue stained MCF-7 target cells were incubated with T cells from individual conditioned DC cocultures as before, as well as control T cells at ratios of 1:5 (E:T) for 4 h. Cytotoxicity was calculated compared with MCF-7 cultured without T cells. Data are means + SEM, representing five individual donors and experiments. (D) CellTracker Blue stained MCF-7 (black bars) or T47D (white bars) breast carcinoma cells were incubated with T cells from individual cocultures at a 1:5 ratio (E:T) for 4 h prior to cytotoxicity measurements. Data are means + SEM, representing four individual donors and experiments. *p < 0.05; ANOVA with Bonferroni's correction.

Download figure to PowerPoint

Tumor cell supernatant primed or unstimulated DCs were cocultured with IL-2-enriched autologous PBMCs for 3 days (Fig. 1A). IL-2-enriched PBMCs were mainly T cells (mononuclear phagocytes, B cells or NK cells <1%, Supporting Information Fig. 2). Lymphocytes derived from these cocultures were then added to living CellTracker Blue (Invitrogen, CA) stained MCF-7 cells for 4 h at different ratios and MCF-7/lymphocyte cocultures were assessed for tumor-cell death. Specific cytotoxicity was not observed up to a ratio of one tumor cell to two T cells (1:2) and reached a plateau at a ratio of 1:5 (Fig. 1B). At this ratio, the VCM group unexpectedly showed significantly higher cytotoxicity toward living MCF-7 cells compared with the control group, whereas T cells from the NCM group were not cytotoxic (Fig. 1C). In contrast, cytotoxicity was reduced below controls when T cells from the ACM group were used, comparable with the basal cytotoxicity exhibited by IL-2 activated T cells alone (Fig. 1C). Importantly, VCM-induced cytotoxicity was cell specific, since alterations in cytotoxicity were not observed when lymphocytes from MCF-7 supernatant-primed DC cocultures where added to T47D cells (Fig. 1D).

image

Figure 2. Apoptotic cell supernatants induce CD69-expressing Treg cells. (A–F) The profile of human T -cell subpopulations from 3-day cocultures with unprimed, ACM-, VCM- or NCM-primed human monocyte-derived autologous DCs. (A) Representative flow cytometry traces are displayed. CD3+T cells subclassed according to CD4 and CD8 expression. CD4+T cells were analyzed for expression CD25+ versus FoxP3+. CD4+CD25+FoxP3+ cells were subdivided based on expression of CD39 and CD69. (B) Statistical quantitation of CD4+ versus CD8+ cells as percentage of CD3+T cells. Data are displayed as mean + SEM, representing five individual donors and experiments. (C) Statistical quantification of FoxP3-expressing CD4+CD25+T cells (Treg cells). Gates were created as in (A). Each point represents one donor and the means of seven individual donors are shown. (D–F) Quantification of Treg-cell types 3 days after coculture with unprimed, ACM-, VCM- or NCM-primed human monocyte-derived autologous DCs. Gates were created as shown in (A). Each point represents one donor and the means of seven individual donors are shown. (D) Statistical quantification of CD39+ and CD69Treg cells as a percentage of CD4+CD25+FoxP3+ cells, (E) CD39CD69+Treg cells as a percentage of CD4+CD25+FoxP3+ cells, (F) CD39+CD69+Treg cells as a percentage of CD4+CD25+FoxP3+ cells. *p < 0.05, **p < 0.01; ANOVA with Bonferroni's correction.

Download figure to PowerPoint

CD39/CD69-expressing FoxP3+Treg cells accumulate in ACM-primed cocultures

Since ACM-priming of DCs suppressed cytotoxicity compared with the VCM- or NCM-primed groups, we determined alterations in the T-cell populations that would account for suppression of tumor killing using polychromatic flow cytometry (Fig. 2A). CD3+ T-cell numbers (not shown) and the ratio of CD4+ versus CD8+ T cells (Fig. 2B) remained unchanged throughout the experimental groups. The same was true for the relative amount of CD4+CD25+FoxP3+ Treg cells (Fig. 2C). Next, we analyzed expression of the ectonucleotidase CD39, found on naturally occurring FoxP3+ Treg cells [[15]], and the lymphocyte activation marker CD69, expressed by Treg cells of cervical cancer patients [[16]], by CD4+CD25+FoxP3+ cells. Expression of CD39 by Treg cells was similar between the coculture setups (Fig. 2D), whereas CD69 was upregulated on Treg cells selectively in the ACM group (Fig. 2E), most significant in the population coexpressing CD39 (Fig. 2F). This regulation pattern was Treg-cell specific, since neither CD39 nor CD69 were significantly upregulated in the total CD4+CD25+ population (Supporting Information Fig. 3A). CD39 was usually not expressed by CD8+ T cells. However, in approximately 20% of all donors, a small subpopulation of CD8+ T cells expressed CD39, which was selectively elevated in the ACM group (Supporting Information Fig. 3B). Thus, only ACM-primed DCs induced surface CD69 expression in cocultured CD39+ Treg cells.

image

Figure 3. Treg cells confer ACM-induced suppression of cytotoxicity. (A) Treg cells were isolated from 2-day cocultures of ACM- or VCM-primed human monocyte-derived autologous DCs using automated magnetic positive selection. Residual T cells were added back to cocultures for 24 h. MCF-7 cells were then incubated with T cells from the ACM or VCM groups with or without (Dpl) Treg cells, as well as controls, and cytotoxicity was analyzed. Data are means + SEM, representing five individual donors and experiments. (B) Cytotoxicity assay: Treg cells were isolated from 2-day cocultures of ACM- or VCM-primed human monocyte-derived autologous DCs as before. Treg cells from ACM groups were then mixed with Treg-cell-depleted T cells from VCM groups and vice versa and added back to the cocultures for 24 h. MCF-7 cells were subsequently incubated with T cells from the Treg-cell exchange groups (Ex) compared with nonexchanged groups and the control. T cells cultured without DCs are indicated as control T. Data are means + SEM, representing six individual donors and experiments. (C and D) CD39+ cells were depleted from IL-2 enriched PBMCs by automated magnetic bead sorting using anti-FITC microbeads after labeling with CD39-FITC antibody. CD39-depleted (CD39 Dpl) T cells were added to cocultures in the same ratio as unmodified T cells. (C) Representative flow cytometry traces displaying the proportion of CD4+CD25+FoxP3+T cells, which are CD39+CD69+ in ACM cocultures of CD39-depleted and unmodified T cells. Data are representative of four experiments. (D) MCF-7 cells were incubated with T cells from the VCM group, the ACM group with or without CD39+T -cell depletion (Dpl), as well as controls, and cytotoxicity was analyzed. Data are means + SEM, representing four individual donors and experiments. *p < 0.05, **p < 0.01, ***p < 0.001; ANOVA with Bonferroni's correction.

Download figure to PowerPoint

Next, we asked if the accumulation of CD69-expressing Treg cells in the ACM group (Fig. 2A and F) contributed to reduced MCF-7 cell killing (Fig. 1B). In a first approach, we depleted Treg cells from unprimed, ACM-primed or VCM-primed cocultures before subjecting the remaining cells to the cytotoxicity assay. Of the isolated CD4+CD25+ T cells, 40% were CD39+FoxP3+ Treg cells (Supporting Information Fig. 4). Interestingly, Treg-cell-depleted lymphocytes from ACM cocultures were significantly more cytotoxic compared with the complete lymphocyte fraction, whereas Treg-cell depletion from VCM-primed cocultures did not affect the enhanced cytotoxicity (Fig. 3A). Next, we asked whether the Treg-cell-dependent suppression of cytotoxicity in the ACM group was transferable. We isolated Treg cells from ACM-primed or VCM-primed cocultures on day 2 and added the Treg cells from the ACM group to the Treg-cell-depleted lymphocytes of the VCM group and vice versa. After 24 h of coincubation, these mixed populations were used in the cytotoxicity assay. Treg cells from the ACM group significantly suppressed cytotoxicity in the VCM group, but Treg cells from the VCM group were unable to suppress cytotoxicity in the ACM group (Fig. 3B). Thus, Treg cells from ACM cocultures suppressed cytotoxicity, correlated to coexpression of CD39 and CD69. We further explored this correlation by depleting CD39+ cells before adding IL-2-enriched lymphocytes to DC cocultures. T cells from these cocultures lacked Treg cells, especially the CD39+ subpopulation, indicating that CD39+ Treg cells upregulate CD69 expression in ACM cocultures, instead of CD69+ cells upregulating CD39 (Fig. 3C). Importantly, depletion of CD39+ T cells restored cytotoxicity in the ACM group as observed when depleting CD25+ cells (Fig. 3D).

image

Figure 4. S1PR4 on DCs conveys ACM-dependent suppression of cytotoxicity. (A–D) T cells were cocultured with control or ACM-primed autologous DCs with or without the S1PR2/4 antagonist JTE-013 (JTE) or the S1PR1/3 antagonist VPC23019 (VPC) for 3 days. (A) Cytotoxicity of T cells from individual cocultures toward living MCF-7 tumor cells. Data are means + SEM, representing six individual donors and experiments. (B) Representative flow cytometry traces of CD39 and CD69 expression by Treg cells from the individual cocultures. (C) CD39+CD69+Treg cells or (D) CD39CD69+Treg cells quantified from flow cytometry staining as a proportion of CD4+CD25+FoxP3+ cells. Each point represents one donor and the means of six individual donors are shown. (E–G) T cells were cocultured with control (nonprimed) or ACM-primed autologous DCs with or without the S1PR4 antagonists CYM74 or CYM58. (E) Cytotoxicity induced by T cells from individual cocultures toward living MCF-7 tumor cells. Data are presented as means + SEM, representing four individual donors and experiments. (F) Representative flow cytometry plots of CD39 and CD69 expression on CD4+CD25+FoxP3+T cells from the cocultures. (G) CD39+CD69+Treg cells gated as in (F) as a proportion of CD4+CD25+FoxP3+T cells from individual cocultures. Each point represents one donor and the means of four individual donors are shown. (H and I) T cells were cocultured with control, ACM- or NCM-primed autologous DCs with or without adding 1 μM S1P. (H) Cytotoxicity induced by T cells from individual cocultures toward living MCF-7 cells. Data are means + SEM, representing six individual donors and experiments. (I) CD39+CD69+Treg cells as a percentage of CD4+CD25+FoxP3+T cells from individual cocultures. Each point represents one donor and the means of six individual donors are shown. *p < 0.05, **p < 0.01, ***p < 0.001; ANOVA with Bonferroni's correction.

Download figure to PowerPoint

S1P in ACM confers suppression of cytotoxicity by activating S1PR4 on DCs

Next, we interfered with the immunosuppressive properties of ACM to clarify whether CD69 expression on FoxP3+ T cells accounted for reduced cytotoxicity. We asked for the factor(s) in ACM inducing DC-dependent suppression of cytotoxicity. Among the immunomodulatory factors secreted by ACs is the lipid mediator S1P (∼10 nM in ACM), determined routinely [[17]]. S1P couples to five specific receptors (S1PR). Human DCs express S1PR1-4. Pharmacological inhibition of S1PRs during ACM priming of human DCs was used to test an impact of AC-derived S1P on DC-dependent T-cell activation. JTE-013, an inhibitor of S1PR2 (IC50 1.5 μM) and S1PR4 (IC50 4.5 μM) [[18]], significantly prevented ACM-induced suppression of cytotoxicity at 15 μM, whereas the S1PR1/3 inhibitor VPC23019 (1 μM) did not (Fig. 4A). Furthermore, JTE-013 significantly reduced the accumulation of CD39+CD69+ Treg cells, whereas the relative proportion of CD69+CD39 Treg cells was unchanged (Fig. 4B–D). To finally identify the S1PR subtype, we used the specific S1PR4 antagonists CYM50374 and CYM50358 (200 nM each) [[19]]. Both substances reversed suppression of cytotoxicity induced by ACM priming (Fig. 4E) and decreased the expansion of CD39+CD69+ Treg cells (Fig. 4F and G). Moreover, supplying S1P (1 μM) during VCM-priming of DCs suppressed the VCM-induced cytotoxicity (Fig. 4H) and increased the number of CD39+CD69+ Treg cells (Fig. 4I). These findings indicate that S1PR4 activation by S1P in ACM enabled DCs to induce CD69 expression on Treg cells, correlating to suppressed cytotoxicity.

ACM-primed DCs secrete IL-27 to activate suppressive Treg cells

Tumor-associated DCs exist in different functional states depending on the microenvironment they are exposed to. We checked DC functional parameter alteration by ACM. Regarding maturation markers, each tumor supernatant slightly induced HLA-DR (human leukocyte antigen-D related) expression on DCs compared with control DCs. HLA-ABC, CD80, and CD83 expression remained largely unchanged, whereas CD86 and CD40 expression were strongly induced with all conditioned media (Supporting Information Fig. 1B). Thus, immunosuppression by ACM was independent of altered maturation.

Functional markers of tolerogenic DCs include proteins such as indolamine-2,3-dioxygenase (IDO). IDO promotes tolerance through depleting tryptophan, which halts T-cell proliferation and/or by accumulation of 3-hydroxykynurenine or 3-hydoxyanthranilic acid, which is toxic to lymphocytes. IDO is expressed by DCs interacting with ACs [[20]]. However, we found that neither inhibition of IDO1 using D-1MT nor IDO2 with L-1MT was able to significantly restore cytotoxicity in the ACM group compared with the VCM group (Supporting Information Fig. 5), although L-1MT mildly elevated cytotoxicity in some experiments.

image

Figure 5. ACM induces IL-27 in DCs in a S1PR4-dependent manner to generate Treg cells. (A–C) Human monocyte-derived DCs were untreated (controls) or treated with VCM, NCM, or ACM with or without the addition of JTE-013 (15 μM) or VPC23019 (1 μM) for 16 h. (A) Secreted cytokines (IL-6, IL-10, IL-12, TNF-α) in DC supernatants were quantified using human inflammatory cytokine Cytometric Bead Arrays. Each point represents one donor and the means of four to six individual donors are shown. (B) Relative mRNA expression of ebi3, p28, and p35 in DCs measured via quantitative PCR. Data are means + SEM, representing four individual donors and experiments. (C) IL-27 protein secretion by DCs as measured by ELISA. Each point represents one donor and the means of six individual donors are shown. (D–F) T cells were cocultured with nonprimed (C), VCM- or ACM-primed autologous DCs with or without the addition of an IL-27-neutralizing antibody (α-IL-27) or the isotype control (IgG). (D) Cytotoxicity induced by T cells from individual cocultures toward living MCF-7 cells. Data are means + SEM, representing seven individual donors and experiments. (E) Representative flow cytometry traces of CD39 and CD69 expression by CD4+CD25+FoxP3+T cells. (F) The percentage of CD4+CD25+FoxP3+T cells coexpressing CD39+ and CD69+ is shown. Each point represents one donor and the means of seven individual donors are shown. (G–I) MCF-7 cells were subjected to 0.5 μg/mL staurosporine (STS) or 30 μM oxaliplatin (OXA) to induce cell death and supernatants were harvested. (G) Expression of ebi3 mRNA in DC treated with STS-ACM or OXA-ACM compared with control DCs is displayed. Data are means + SEM, representing four individual donors and experiments. (H) T cells were cocultured with nonprimed (C), VCM-, STS-ACM - or OXA-ACM -primed autologous DCs. Representative flow cytometry traces of CD39 and CD69 expression by Treg cells from the individual cocultures are shown (n = 5). (I) Cytotoxicity induced by T cells from individual cocultures toward living MCF-7 cells. Data are means + SEM, representing five individual donors and experiments. *p < 0.05, **p < 0.01, ***p < 0.001; ANOVA with Bonferroni's correction.

Download figure to PowerPoint

Next, we measured the release of inflammatory cytokines 16 h after stimulation of DCs with tumor-cell supernatants in combination with S1PR antagonists. IL-12 was not secreted in relevant amounts (Fig. 5A). IL-6 showed a trend toward enhanced production in the VCM group, which was significant for TNF-α. However, secretion of these cytokines was enhanced when using VPC23019 together with ACM, but not JTE-013, not correlating with changes in cytotoxicity. Release of IL-10 was interestingly elevated only with ACM and ACM+VPC23019 (Fig. 5A), although being in a low pg/mL range largely restricts its potential impact.

Next, we focused on IL-12 family cytokines, since despite sharing subunits, these cytokines enhance generation of either Th1, Th17, or Treg cells [[21]]. For instance, the protein encoded by Epstein-Barr virus induced gene 3 (ebi3) is a common subunit of IL-35 and IL-27. We noticed that ebi3 mRNA was upregulated with ACM after 16 h in a S1PR2/4-dependent manner (Fig. 5B). Since expression of the complementary subunits for IL-27 or IL-35, p28, and p35, were unchanged with ACM (Fig. 5B), we analyzed secretion of IL-35 and IL-27 using ELISA. While detecting IL-35 was unsuccessful, IL-27 was significantly upregulated in ACM-treated DCs compared with controls or VCM-primed DCs, which was abolished when inhibiting S1PR2/4 (Fig. 5C). To check for IL-27-dependent generation of suppressive Treg cells, we added a specific IL-27-neutralizing antibody versus the isotype control (each 1 μg/mL) to DCs before adding autologous T cells. Blocking IL-27 potently reduced ACM-induced suppression of cytotoxicity compared with the isotype control (Fig. 5D) and reduced ACM-induced CD69 expression in CD39+ Treg cells (Fig. 5E and F).

We wondered whether induction of immunogenic cell death would induce IL-27-dependent CD39+CD69+ Treg-cell expansion. Comparing the influence of oxaliplatin-treated MCF-7 cell supernatants (OXA-ACM) with staurosporine-treated cells (STS-ACM) on DCs, we noticed significantly lower ebi3 expression in OXA-ACM-treated DCs (Fig. 5G). Accordingly, CD69+CD39+ Treg cells in OXA-ACM cocultures were reduced compared with STS-ACM cocultures (Fig. 5H), resulting in enhanced cytotoxicity of T cells from OXA-ACM cocultures compared with T cells from the STS-ACM group (Fig. 5I). Hence, oxaliplatin-induced immunogenic cell death did not result in expansion of suppressive CD69+CD39+ Treg cells, likely due to reduced IL-27 secretion from DCs.

Suppression of cytotoxicity depends on adenosine generation

We were interested in mechanisms how Treg cells suppressed cytotoxicity. Analyzing the contents of the T-cell-derived cytokines IFN-γ, IL-10, IL-4, IL-17, IL-2 from total cocultures after day 3, did not reveal any meaningful regulation patterns (Supporting Information Fig. 6). Treg cells can suppress cytotoxic T cells by various mechanisms including secretion of TGF-β and IL-10 [[22]]. However, when we analyzed IL-10 mRNA expression in isolated Treg cells or TGF-β expression in total cocultures (by intracellular staining), unexpectedly neither IL-10 (Fig. 6A) nor TGF-β expression (Fig. 6B) was altered in Treg cells upon ACM-stimulation. Furthermore, neutralizing TGF-β in ACM cocultures with a specific antibody did not restore cytotoxicity (Fig. 6C).

image

Figure 6. Interfering with adenosine generation and signaling restores cytotoxicity. (A) Magnetic bead isolated Treg cells from 3-day cocultures (untreated (C)) or treated with ACM, VCM, or NCM with or without the addition of 15 μM JTE-013) were analyzed for IL-10 mRNA expression relative to the control via real-time quantitative polymerase chain reaction (qPCR). Data are means + SEM, representing five individual donors and experiments. (B) TGF-β expression in CD4+CD25+FoxP3+Treg cells, from the cocultures described in (A) was determined via intracellular staining and polychromatic flow cytometry and the mean fluorescence intensity is displayed. Data are means + SEM, representing four individual donors and experiments. (C) T cells were cocultured with nonprimed DCs (C), VCM- or ACM-primed autologous DCs with or without the addition of a TGF-β-neutralizing antibody (α-TGF-β) or the isotype control (IgG). Cytotoxicity induced by T cells from individual cocultures toward living MCF-7 cells was determined. Data are means + SEM, representing five individual donors and experiments. (D) Flow cytometry traces indicating CD39 expression by CD8T cells from cocultures with nonactivated DCs (control). These cells were gated and evaluated for FoxP3 versus CD73 expression. (E) Relative flow cytometry traces indicating CD73 expression by CD8+Tcells from cocultures with nonactivated DCs (control). These cells were gated and evaluated for FoxP3 versus CD39 expression. (F) Percentage of CD8+T cells which expressed CD73 following the gating strategy shown in (E). Each point represents one donor and the means of seven individual donors are shown. (G) T cells from VCM cocultures were preincubated for 1 h with anti-CD8 or the respective isotype control. These T cells were used to determine cytotoxicity against living MCF-7 cells as compared with T cells from control cocultures. Data are shown as mean + SEM representing five individual donors and experiments. (H) T cells were cocultured with control, VCM- or ACM-primed autologous DCs with or without addition of inhibitor against CD39 (ARL; 250 μM), CD73 (APCP; 100 μM) or adenosine receptor A2a (CSC; 10 mM). Cytotoxicity induced by T cells from individual cocultures toward living MCF-7 cells is shown. Data are means + SEM, representing six individual donors and experiments. (F) Quantification of CD73 expression by CD8+T cells from individual cocultures as before, plus cocultures of ACM-primed DCs with anti-IL-27 (1 μg/mL), analyzed using flow cytometry, is displayed. Each point represents one donor and the means of four individual donors are shown. *p < 0.05, **p < 0.01, ***p < 0.001; ANOVA with Bonferroni's correction.

Download figure to PowerPoint

Another molecule known for its immunosuppressive function is adenosine, produced by the sequential breakdown of ATP by, for example, the ectonucleotidases CD39 and CD73. Extracellular adenosine inhibits proliferation and/or priming of CD8+ T cells [[23]], which, as CD8 depletion experiments suggested, were required for VCM-induced cytotoxicity (Fig. 6D). CD39 is expressed by human Treg cells, whereas CD73 might not be coexpressed by these cells. Indeed, CD39 was mainly expressed by CD4+ Treg cells, which did not express CD73 (Fig. 6C). CD73 was only expressed on CD8+ cells (Fig. 6D), which was unaltered in cocultures (Fig. 6E). Nevertheless, adenosine was involved in ACM-induced suppression of cytotoxicity. Addition of the CD39 inhibitor ARL67156, the CD73 inhibitor APCP [[24]] as well as the adenosine receptor A2a inhibitor CSC [[25]] restored cytotoxicity in ACM cocultures (Fig. 6F). None of the compounds altered expression of CD39 by Treg cells or CD73 by CD8+ T cells except for the CD73 inhibitor APCP, which unexpectedly diminished CD73 expression on CD8+ T cells (Fig. 6G).

Our data suggested that CD69 expression by CD39+ Treg reduced cytotoxicity. However, a concerted action of CD39 and CD73 was important for suppression of cytotoxicity induced by ACM priming, although these molecules were expressed on different cells. CD69 is a member of the C-type lectin family, proteins which regulate cell–cell contact. We hypothesized that CD69-bearing CD39+ Treg cells might establish direct contact with CD73-expressing CD8+ T cells to ensure efficient adenosine generation. To approach this question, we analyzed CD4+CD8+ events within CD3+ doublet events from control, ACM and VCM cocultures using polychromatic flow cytometry (Supporting Information Fig. 7). These CD4+CD8+ doublets were generally enriched in CD25+CD69+FoxP3+ cells (Supporting Information Fig. 7). However, this enrichment was strongly pronounced in the ACM group compared with the control or VCM groups (Fig. 7A and B), together with a significant enrichment of CD4+CD8+ events within whole CD3+ doublets in the ACM group (Fig. 7C). This pattern was also observed when analyzing whether CD25+CD39+CD69+ events were over-represented in doublets compared to the singlet population (Fig. 7D). Naturally, we wondered whether antibody-mediated CD69 depletion would reduce CD4+CD8+ doublet formation as well as enrichment of CD25+CD39+CD69+ events in doublets of the ACM group. CD69 depletion was efficient, without altering the total amount of CD39+ cells (Fig. 7E). Strikingly, CD4+CD8+ doublets (Fig. 7F) as well as CD25+CD39+ events in the doublet population (Fig. 7G) of the ACM group were decreased upon CD69 neutralization. These findings provide a first hint that CD69-expressing Treg cells may bind to an unidentified ligand on CD8+ T cells, thus aiding adenosine production and subsequent suppression of cytotoxicity.

image

Figure 7. CD39/CD69-expressing Treg cells are enriched in CD4+CD8+ doublet events. (A) Representative flow cytometry traces show analysis of the FSC-W gated CD3+ doublet population of cells from control, ACM- or VCM-primed cocultures. CD4+CD8+ events, most likely aggregates of CD4+ and CD8+T cells, were analyzed for expression of CD25, FoxP3, CD39, and CD69. (B) Quantification of the percentage of CD25+FoxP3+CD39+CD69+ events within CD4+CD8+ events as gated in (A) is shown. (C) Quantification of the percentage of CD4+CD8+ events in the CD3+ doublet population. (D) The ratio of CD25+FoxP3+CD39+CD69+ events in the CD4+CD8+CD3+ doublet population versus the CD4+CD3+ single-cell population in ACM-, VCM-, or control cocultures is shown. (B–D) Each data point represents one donor and the means of seven individual donors are shown. (E) ACM cocultures were treated with either anti-CD69 or an isotype control (IgG) on day 2 of culture. Representative flow cytometry plots (n = 6) show CD39 and CD69 expression on CD4+T cells on day 3 of culture. (F) Quantification of the percentage of CD4+CD8+ events in the CD3+ doublet population in ACM-cocultures with anti-CD69 (α-CD69) or an isotype control (IgG) on day 3 of culture is shown. (G) The ratio of CD25+FoxP3+CD39+CD69+ events in the CD4+CD8+CD3+ doublet population versus the CD4+CD3+ single-cell population of ACM cocultures with anti-CD69 (α-CD69) or an isotype control (IgG) on day 3 of culture. (F and G) Each data point represents one donor and the means of seven individual donors are shown. *p < 0.05, **p < 0.01, ***p < 0.001; (B–D) ANOVA with Bonferroni's correction or (F and G) paired Student's t-test.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Reagents
  8. Acknowledgements
  9. Conflict of interest
  10. References
  11. Supporting Information

A growing tumor or a tumor subjected to conventional therapy sheds tumor-derived factors/exosomes [[26]]. In this study, factors shed from apoptotic tumor cells reduced cytotoxicity, whereas priming with VCM increased cytotoxicity against living tumor cells. We propose that antigen-containing exosomes in VCM might be responsible for inducing cytotoxicity via CD8+ T cells, whereas Treg cells induced by immunosuppressive factors in ACM prevent cytotoxicity. However, tumor cell specific CTLs were still generated by ACM as indicated by Treg-cell depletion or transfer experiments. Hence, apoptotic debris resulting from cytotoxic cancer therapy, which is known to induce immune paralysis [[5]] when shed to the TDLNs might induce generation of Treg cells that block CTL activity. Along this line, depletion of Treg cells using anti-CD25 or anti-CTLA4 along with T-cell or DC immunotherapy restored antitumor immunity [[4]].

Generation of suppressive Treg cells required activation of S1PR4 on DCs, likely due to S1P that is secreted by ACs [[17]]. S1P shifts LPS-induced maturation of DCs from Th1 to Th2 by suppressing IL-12 release and elevating IL-4 and IL-10 production [[27]]. We observed that S1PR1/3 inhibition upon ACM treatment indeed marginally increased secretion of IL-12, TNF-α, and IL-6, while ACM moderately increased IL-10 (Fig. 5A). This suggests that S1P acting on S1PR1/3 might be an intrinsic attenuating signal in AC-induced inflammation. However, for suppression of cytotoxicity S1P in ACM rather induced IL-27 release through S1PR4 (Fig. 5B and C). Interestingly, expression of IL-27 by APCs in the lymphatic system upon interaction with tumor cells has been demonstrated before [[28]].

The function of IL-27 in T-cell biology is ambiguous, varying between inflammatory (induction of Th1) and immunosuppressive [[29]]. IL-27 induces expression of the Th1 transcription factor T-bet [[30]] and promotes CD8+ T-cell proliferation [[31]] by activating STAT1. However, also Treg cells can express T-bet. These specialized Treg cells migrate to areas of Th1 inflammation and contribute to dampening overactivation of immunity. A similar mechanism may be employed by ACM-primed DCs expressing IL-27 to induce Treg cells that in turn suppress cytotoxicity. Besides inducing T bet, IL-27 potently reduces expression of the Th17 cell-determining transcription factor RORC, but not Foxp3 [[32]]. In our hands, IL-27 induced CD69 expresssion in CD39+ Treg cells. The transcriptional program involved remains to be discovered.

The exact mechanism employed by Treg cells to suppress cytotoxicity in our system is yet unclear. The defining characteristic of the suppressive Treg-cell subpopulation expanded by ACM-primed DCs was the expression of both CD39 and CD69. As suggested previously, CD39 was mainly expressed by FoxP3+ cells [[15]]. CD39 metabolizes ATP and ADP to AMP, the former being produced by activated T cells [[33]]. Since DCs treated with ACM do not show deficiencies in maturation or activation, they might well engage the TCR of T-effector cells to stimulate ATP release, which can be metabolized to AMP by CD39+ Treg cells, and further degraded to adenosine by CD73, present exclusively on CD8+ T cells in our system. Extracellular adenosine may inhibit many aspects of T-cell function such as effector differentiation, activation, cytokine production, metabolic activity, and proliferation [[23]]. Interestingly, ectonucleotidase expression and activity was increased in Treg cells of head and neck cancer patients [[24]]. Ectonucleotidase can also be expressed by ovarian carcinoma cells, which generate adenosine to inhibit CD4+ T-cell proliferation as well as NK-cell cytotoxicity through activation of AdorA2a on these cells [[34]]. Hence, adenosine generated through CD39 and CD73 expressed by Treg cells and CD8+ T cells, respectively, might suppress the function of CTLs in our system, acting via AdorA2a, whose inhibition restored cytotoxicity in the ACM group.

Besides CD39, we hypothesize that CD69 on Treg cells might directly suppress the activity of effector T cells. CD69 is a C-type lectin, which can trigger TGF-β production [[35]]. Although TGF-β was connected with suppression of cytotoxic CD8+ T cells previously [[36]], we did not observe significant upregulation of TGF-β in Treg cells and TGF-β neutralization did not restore cytotoxicity. An alternative option might be binding of CD69 to a putative “ligand/receptor” on the surface of CD8+ T cells, which is common for C-type lectins, for example, for interactions between cytotoxic lymphocytes and their targets [[37]]. However, a binding partner for CD69 is not known. If this putative molecule is expressed on CD8+ T cells, CD69-expressing Treg cells might bind to these cells, creating a functional platform for adenosine production by bringing CD39 and CD73 in close proximity. Our analysis of CD4+CD8+ doublets supports this hypothesis. Future experiments addressing the function and the putative ligand for CD69 are needed.

The efficacy of chemotherapy may also depend on its impact on the immune system. Chemotherapeutics may either kill tumor cells directly, which may or may not be immunogenic, may cause tumor-cell death by activating immune cells or cause immunosuppression by also killing immune cells [[38]]. The immunological outcome depends on surface alterations of dying cells or, as seen in our system, on the AC secretome, which depends on the cell death inducing agent. In our studies oxaliplatin, which is known as an immunogenic cell death inducing agent, did indeed not suppress cytotoxicity.

Ex vivo priming of DCs with tumor lysates and in vivo DC activ-ation strategies have been employed in cancer immunotherapy [[39]]. Our data add to the understanding how priming with viable or killed tumor cells affects DC biology and thus might be valuable regarding strategies of ex vivo DC activation. Also, conventional therapy might benefit from inhibition of intrinsic immunosuppressive pathways. Our results suggest that interfering with S1PR4 and/or IL-27 might restrict tumor-induced immune suppression.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Reagents
  8. Acknowledgements
  9. Conflict of interest
  10. References
  11. Supporting Information

Primary human immune cell isolation and expansion

PBMCs were obtained from Buffy Coats (DRK-Blutspendedienst Baden-Württemberg-Hessen, Frankfurt, Germany) using Ficoll-Isopaque (PAA, Cölbe, Germany) gradient centrifugation. CD14+ monocytes were isolated from PBMCs by magnetic sorting using CD14 microbeads and the autoMACS™ Separator (Miltenyi, Bergisch Gladbach, Germany). The negative fraction was used for T-cell enrichment in T-cell medium [[40]] containing IL-2 (100 U/mL) (Immunotools, Friesoythe, Germany) for 6 days.

Monocyte-derived DC generation

A total of 2 × 105 human primary monocytes were cultured in 12-well plates in RPMI 1640 containing 10% FCS, GM-CSF (100 ng/mL; Miltenyi) and IL-4 (5 ng/mL) (Immunotools) for 6 days to generate DCs.

Preparation of tumor-cell supernatants

MCF-7 human breast carcinoma cells were grown in RPMI 1640 with 10% FCS. Supernatants of living (VCM), apoptotic (ACM), or necrotic (NCM) MCF-7 cells were prepared as follows. MCF-7 cells remained untreated (living), were exposed to 0.5 μg/mL staurosporine (Sigma, Steinheim, Germany) for 1 h (apoptosis) or 30 μM oxaliplatin (Sigma) for 16 h (immunogenic cell death) or were incubated at 56°C for 30 min (necrosis), washed and incubated for another 5 h in full medium. Conditioned media were harvested by centrifugation (1000 × g, 10 min) and filtration through 0.2 μm pore filters.

Reagents

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Reagents
  8. Acknowledgements
  9. Conflict of interest
  10. References
  11. Supporting Information

S1P and VPC23019 (1 μM; Avanti Polar Lipids, AL, USA) were dissolved following the manufacturer's instructions. JTE-013 (15 μM) (Biomol, Hamburg Germany) and CYM50358 and CYM50374 (each 200 nM) [[19]] were dissolved in DMSO. DCs were preincubated with these reagents for 30 min before adding tumor-cell supernatants. The IDO inhibitors L-1MT or D-1MT (1 mM; Sigma) [[41]] were added to DCs 2 h before T-cell coculture. IL-27 neutralizing antibody and isotype control (R&D Systems, Wiesbaden-Nordernstadt, Germany) were added at 1 μg/mL to DCs 30 min before adding T cells. ARL67156 (250 μM), 5′-(αβ-methylene) diphosphate (APCP; 100 μM) in ddH2O and 8 (3-chlorostyryl) caffeine (CSC) (10 mM) in DMSO, as well as CD69 antibody (BD Biosciences, Heidelberg, Germany), TGF-β neutralizing antibody (R&D Systems) [[42]] and respective isotype controls, were added to DC-T-cell cultures at day 2.

DC–T-cell coculture

Tumor-cell supernatants were added to 2 × 105 DCs at ratios of 1:1 for 16 h, followed by washing. Afterwards 2 × 106 T-cell-enriched PBMCs were added and cocultures were maintained for 3 days.

Cytotoxicity assay

A total of 5 × 104 human breast carcinoma cells (MCF-7, T47D), prestained with 100 μM CellTracker Blue, were cultured for 4 h in flow cytometry tubes with T cells from DC cocultures (ratios as indicated). The reaction mix was stained with propidium iodide (PI) for 10 min. Directly before sample acquisition, Flow-Count Fluorospheres (Beckman-Coulter, Krefeld, Germany) were added as an internal cell counting standard and 2000 living (PI, CellTracker Blue+, FSChigh) breast cancer cells were recorded for each sample. Cytotoxicity was calculated as described [[43]]. Blocking of cytotoxic T cells was performed using CD8 antibody [[44]] or the respective isotype control (BD Biosciences) 1 h before starting cytotoxicity experiments.

Treg-cell and CD39+-cell isolation

Treg cells were isolated from DC-T-cell cocultures using CD4+CD25+ Treg isolation Kits (Miltenyi). Treg-cell-depleted populations were added back to the respective cocultures. Isolated Treg cells (controlled via flow cytometry, Supporting Information Fig. 3) were either used for RNA isolation or were interchanged between ACM and VCM groups at ratios reconstituting mean FoxP3-expressing cells (0.5%) to monitor their specific suppressive potential. CD39-expressing T cells from IL-2 enriched T-cell cultures were removed by staining with CD39-FITC antibody (Miltenyi) and magnetic separation using anti-FITC microbeads (Miltenyi). CD39-depleted populations were then used for cocultures.

Flow cytometry

For analysis of DC maturation, DCs were harvested using accutase (PAA), and stained for 20 min with CD86-FITC (Immunotools), HLA-DR-PE-Cy7 (BD Biosciences) or HLA-ABC (MHC I)-FITC (Miltenyi) CD80-PE, CD83-allophycocyanin, CD40-FITC (BioLegend, San Diego, CA, USA). For polychromatic flow cytometry analysis of cocultures, nonspecific antibody binding to FC-γ receptors was blocked using Human Fc Receptor Binding Inhibitor (eBioscience, San Diego, CA, USA) for 20 min, cells were resuspended in FACS staining buffer (BD Biosciences) and incubated with the following antibodies: CD3-V450, CD4-V500, CD8-allophycocyanin-H7, CD25-PE-Cy7, CD69-AlexaFlour 700, and CD73-PE (BD Biosciences), CD39-FITC (Miltenyi), CD19-Qdot 655 (Invitrogen, Carlsbad, CA, USA) on ice for 30 min. Cells were fixed and permeabilized using the FoxP3 buffer set (BD Biosciences) and incubated with FoxP3-APC antibody (BD Biosciences). To analyze TGF-β expression, cells were pretreated with 500 ng/mL Brefeldin A (Sigma) and TGF-β1-PE antibody (IQ products, Groningen, The Netherlands) was used alongside the FoxP3 antibody. Samples were acquired using a LSRII/Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo 7.6.1 (Treestar, Ashland, OR, USA). Antibodies were titrated to determine optimal concentrations. CompBeads (BD Biosciences) were used to create compensation matrices. For gating, fluorescence minus one controls and/or isotype controls was used. Instrument calibration was controlled and adjusted daily using Cytometer Setup and Tracking beads (BD Biosciences).

RNA isolation, cDNA synthesis, and real-time quantitative PCR

RNA from DCs was isolated using PeqGold (Peqlab, Erlangen, Germany) and quantitated using the NanoDrop spectrophotometer (NanoDrop, Wilmington, USA). RNA from <105 Treg cells was isolated using the RNeasy micro kit (Qiagen, Hilden, Germany), quantitated using Bioanalyzer (Agilent, Böblingen, Germany) and transcribed with sensiscript RT kits (Qiagen). Quantitative PCR was performed as described [[42]]. Human ebi3, actin, and 18S rRNA were amplified using QuantiTect Primer Assays (Qiagen, Hilden, Germany). Additional primer sets were p35 sense: 5′-AGATA AAACC AGCACA GTGG AGGC-3′, antisense: 5′-GCC AGGC AACTC CCATT AGT TAT-3′; p28 sense: 5′-AGGA GCT GCGGA GG GAGTT-3′, antisense: 5′-AGGG GCAGG AGGTA CAG GTTC-3′; IL-10 sense: 5′-AAGC CTG ACCA CGCTT TCTA-3′, antisense: 5′-TAGCA GTTAG GAAGCCC CAA-3′. Results were analyzed using Gene Expression Macro (Bio-Rad, München, Germany). Actin and 18S were internal controls.

Cytokine quantitation

TNF-α, IL-10, IL-6, IL-12 concentrations in DC supernatants and IFN-γ, IL-10, IL-4, IL-17, IL-2 from DC/T-cell cocultures were quantified using Human Inflammatory Cytokine or Human Th1/Th2/Th17 kits (BD Biosciences). Samples were acquired by flow cytometry and processed with BD Biosciences FCAP software. IL-27 levels in DC supernatants were quantified using sandwich ELISA (BioLegend).

Statistical analysis

Data were analyzed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). p-Values were calculated using ANOVA with Bonferroni's correction. Differences were considered significant at p < 0.05.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Reagents
  8. Acknowledgements
  9. Conflict of interest
  10. References
  11. Supporting Information

The authors thank Miguel Guerrero and Mariangela Urbano for the S1P4R antagonists CYM74 or CYM58, Franz-Josef Streb and Margarethe Wiebe for excellent technical existence. A. W. was supported by a grant from Medical Faculty, Goethe-University Frankfurt, B.B. is supported by Sander Foundation (2007.070.2) and DFG (Br999, ECCPS). E. R. is supported by a National Institute of Health Molecular Library Screen Center Network grant (U54 MH084512A).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Reagents
  8. Acknowledgements
  9. Conflict of interest
  10. References
  11. Supporting Information
  • 1
    Dhodapkar, M. V., Dhodapkar, K. M. and Palucka, A. K., Interactions of tumor cells with dendritic cells: balancing immunity and tolerance. Cell Death Differ. 2008. 15: 3950.
  • 2
    Piersma, S. J., Welters, M. J. and van der Burg, S. H., Tumor-specific regulatory T cells in cancer patients. Hum. Immunol. 2008. 69: 241249.
  • 3
    Munn, D. H. and Mellor, A. L., The tumor-draining lymph node as an immune-privileged site. Immunol. Rev. 2006. 213: 146158.
  • 4
    Zou, W., Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat. Rev. Cancer 2005. 5: 263274.
  • 5
    Zitvogel, L., Apetoh, L., Ghiringhelli, F. and Kroemer, G., Immunological aspects of cancer chemotherapy. Nat. Rev. Immunol. 2008. 8: 5973.
  • 6
    McDonnell, A. M., Robinson, B. W. and Currie, A. J., Tumor antigen cross-presentation and the dendritic cell: where it all begins? Clin. Dev. Immunol. 2010. 2010: 539519.
  • 7
    Iero, M., Valenti, R., Huber, V., Filipazzi, P., Parmiani, G., Fais, S. and Rivoltini, L., Tumour-released exosomes and their implications in cancer immunity. Cell Death Differ. 2008. 15: 8088.
  • 8
    Gregory, C. D. and Pound, J. D., Cell death in the neighbourhood: direct microenvironmental effects of apoptosis in normal and neoplastic tissues. J. Pathol. 2010. 223: 177194.
  • 9
    Albert, M. L., Death-defying immunity: do apoptotic cells influence antigen processing and presentation? Nat. Rev. Immunol. 2004. 4: 223231.
  • 10
    Zitvogel, L., Kepp, O. and Kroemer, G., Decoding cell death signals in inflammation and immunity. Cell 2010. 140: 798804.
  • 11
    Nowak, A. K., Lake, R. A., Marzo, A. L., Scott, B., Heath, W. R., Collins, E. J., Frelinger, J. A. et al., Induction of tumor cell apoptosis in vivo increases tumor antigen cross-presentation, cross-priming rather than cross-tolerizing host tumor-specific CD8 T cells. J. Immunol. 2003. 170: 49054913.
  • 12
    Morelli, A. E. and Thomson, A. W., Tolerogenic dendritic cells and the quest for transplant tolerance. Nat. Rev. Immunol. 2007. 7: 610621.
  • 13
    Panaretakis, T., Kepp, O., Brockmeier, U., Tesniere, A., Bjorklund, A. C., Chapman, D. C., Durchschlag, M. et al., Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death. EMBO J. 2009. 28: 578590.
  • 14
    van der Most, R. G., Currie, A. J., Robinson, B. W. and Lake, R. A., Decoding dangerous death: how cytotoxic chemotherapy invokes inflammation, immunity or nothing at all. Cell Death Differ. 2008. 15: 1320.
  • 15
    Borsellino, G., Kleinewietfeld, M., Di Mitri, D., Sternjak, A., Diamantini, A., Giometto, R., Hopner, S. et al., Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune suppression. Blood 2007. 110: 12251232.
  • 16
    Battaglia, A., Buzzonetti, A., Baranello, C., Ferrandina, G., Martinelli, E., Fanfani, F., Scambia, G. et al., Metastatic tumour cells favour the generation of a tolerogenic milieu in tumour draining lymph node in patients with early cervical cancer. Cancer Immunol. Immunother. 2009. 58: 13631373.
  • 17
    Weigert, A., Cremer, S., Schmidt, M. V., von Knethen, A., Angioni, C., Geisslinger, G. and Brune, B., Cleavage of sphingosine kinase 2 by caspase-1 provokes its release from apoptotic cells. Blood 2010. 115: 35313540.
  • 18
    Wetter, J. A., Revankar, C. and Hanson, B. J., Utilization of the Tango beta-arrestin recruitment technology for cell-based EDG receptor assay development and interrogation. J. Biomol. Screen 2009. 14: 11341141.
  • 19
    Guerrero, M., Urbano, M., Zhao, J., Crisp, M., Chase, P., Hodder, P., Schaeffer, M. T. et al., Discovery, design and synthesis of novel potent and selective sphingosine-1-phosphate 4 receptor (S1P-R) agonists. Bioorg. Med. Chem. Lett. 2011. 22: 537542.
  • 20
    Williams, C. A., Harry, R. A. and McLeod, J. D., Apoptotic cells induce dendritic cell-mediated suppression via interferon-gamma-induced IDO. Immunology 2008. 124: 89101.
  • 21
    Gee, K., Guzzo, C., Che Mat, N. F., Ma, W. and Kumar, A., The IL-12 family of cytokines in infection, inflammation and autoimmune disorders. Inflamm. Allergy Drug Targets 2009. 8: 4052.
  • 22
    Shevach, E. M., Mechanisms of foxp3+ T regulatory cell-mediated suppression. Immunity 2009. 30: 636645.
  • 23
    Linnemann, C., Schildberg, F. A., Schurich, A., Diehl, L., Hegenbarth, S. I., Endl, E., Lacher, S. et al., Adenosine regulates CD8 T-cell priming by inhibition of membrane-proximal T-cell receptor signalling. Immunology 2009. 128: e728e737.
  • 24
    Mandapathil, M., Szczepanski, M. J., Szajnik, M., Ren, J., Lenzner, D. E., Jackson, E. K., Gorelik, E. et al., Increased ectonucleotidase expression and activity in regulatory T cells of patients with head and neck cancer. Clin. Cancer Res. 2009. 15: 63486357.
  • 25
    Eltzschig, H. K., Thompson, L. F., Karhausen, J., Cotta, R. J., Ibla, J. C., Robson, S. C. and Colgan, S. P., Endogenous adenosine produced during hypoxia attenuates neutrophil accumulation: coordination by extracellular nucleotide metabolism. Blood 2004. 104: 39863992.
  • 26
    Zeelenberg, I. S., van Maren, W. W., Boissonnas, A., Van Hout-Kuijer, M. A., Den Brok, M. H., Wagenaars, J. A., van der Schaaf, A. et al., Antigen localization controls T cell-mediated tumor immunity. J. Immunol. 2011. 187: 12811288.
  • 27
    Rivera, J., Proia, R. L. and Olivera, A., The alliance of sphingosine-1-phosphate and its receptors in immunity. Nat. Rev. Immunol. 2008. 8: 753763.
  • 28
    Larousserie, F., Bardel, E., Pflanz, S., Arnulf, B., Lome-Maldonado, C., Hermine, O., Bregeaud, L. et al., Analysis of interleukin-27 (EBI3/p28) expression in Epstein-Barr virus- and human T-cell leukemia virus type 1-associated lymphomas: heterogeneous expression of EBI3 subunit by tumoral cells. Am. J. Pathol. 2005. 166: 12171228.
  • 29
    Yoshida, H., Nakaya, M. and Miyazaki, Y., Interleukin 27: a double-edged sword for offense and defense. J. Leukoc. Biol. 2009. 86: 12951303.
  • 30
    Kamiya, S., Owaki, T., Morishima, N., Fukai, F., Mizuguchi, J. and Yoshimoto, T., An indispensable role for STAT1 in IL-27-induced T-bet expression but not proliferation of naive CD4+T cells. J. Immunol. 2004. 173: 38713877.
  • 31
    Schneider, R., Yaneva, T., Beauseigle, D., El-Khoury, L. and Arbour, N., IL-27 increases the proliferation and effector functions of human naive CD8+ T lymphocytes and promotes their development into Tc1 cells. Eur. J. Immunol. 2011. 41: 4759.
  • 32
    Murugaiyan, G., Mittal, A., Lopez-Diego, R., Maier, L. M., Anderson, D. E. and Weiner, H. L., IL-27 is a key regulator of IL-10 and IL-17 production by human CD4+T cells. J. Immunol. 2009. 183: 24352443.
  • 33
    Schenk, U., Westendorf, A. M., Radaelli, E., Casati, A., Ferro, M., Fumagalli, M., Verderio, C. et al., Purinergic control of T-cell activation by ATP released through pannexin-1 hemichannels. Sci. Signal. 2008. 1: ra6.
  • 34
    Hausler, S. F., Montalban del Barrio, I., Strohschein, J., Anoop Chandran, P., Engel, J. B., Honig, A., Ossadnik, M. et al., Ectonucleotidases CD39 and CD73 on OvCA cells are potent adenosine-generating enzymes responsible for adenosine receptor 2A-dependent suppression of T-cell function and NK cell cytotoxicity. Cancer Immunol. Immunother. 2011. 60: 14051418.
  • 35
    Sancho, D., Gomez, M. and Sanchez-Madrid, F., CD69 is an immunoregulatory molecule induced following activation. Trends Immunol. 2005. 26: 136140.
  • 36
    Chen, M. L., Pittet, M. J., Gorelik, L., Flavell, R. A., Weissleder, R., von Boehmer, H. and Khazaie, K., Regulatory T cells suppress tumor-specific CD8 T-cell cytotoxicity through TGF-beta signals in vivo. Proc. Natl. Acad. Sci. USA 2005. 102: 419424.
  • 37
    Vogler, I. and Steinle, A., Vis-a-vis in the NKC: genetically linked natural killer cell receptor/ligand pairs in the natural killer gene complex (NKC). J. Innate Immun. 3: 227235.
  • 38
    Zitvogel, L., Kepp, O. and Kroemer, G., Immune parameters affecting the efficacy of chemotherapeutic regimens. Nat. Rev. Clin. Oncol. 2011. 8: 151160.
  • 39
    Le, D. T., Pardoll, D. M. and Jaffee, E. M., Cellular vaccine approaches. Cancer J. 2010. 16: 304310.
  • 40
    Exley, M. A., Wilson, B. and Balk, S. P., Isolation and functional use of human NKT cells. Curr. Protoc. Immunol. 2010. 90: 14.11.114.11.17.
  • 41
    Qian, F., Villella, J., Wallace, P. K., Mhawech-Fauceglia, P., Tario, J. D., Jr., Andrews, C., Matsuzaki, J. et al., Efficacy of levo-1-methyl tryptophan and dextro-1-methyl tryptophan in reversing indoleamine-2,3-dioxygenase-mediated arrest of T-cell proliferation in human epithelial ovarian cancer. Cancer Res. 2009. 69: 54985504.
  • 42
    Herr, B., Zhou, J., Werno, C., Menrad, H., Namgaladze, D., Weigert, A., Dehne, N. et al., The supernatant of apoptotic cells causes transcriptional activation of hypoxia-inducible factor-1alpha in macrophages via sphingosine-1-phosphate and transforming growth factor-beta. Blood 2009. 114: 21402148.
  • 43
    Zimmermann, S. Y., Esser, R., Rohrbach, E., Klingebiel, T. and Koehl, U., A novel four-colour flow cytometric assay to determine natural killer cell or T-cell-mediated cellular cytotoxicity against leukaemic cells in peripheral or bone marrow specimens containing greater than 20% of normal cells. J. Immunol. Methods 2005. 296: 6376.
  • 44
    Campanelli, R., Palermo, B., Garbelli, S., Mantovani, S., Lucchi, P., Necker, A., Lantelme, E. et al., Human CD8 co-receptor is strictly involved in MHC-peptide tetramer-TCR binding and T-cell activation. Int. Immunol. 2002. 14: 3944.
Abbreviations
AC

apoptotic cell

ACM

apoptotic tumor cell condition medium

AdorA2a

adenosine receptor A2a

ebi3

Epstein-Barr virus induced gene 3

NCM

necrotic tumor cell condition medium

OXA

oxaliplatin

S1P

sphingosine-1-phosphate

STS

staurosporine

TDLN

tumor-adjacent draining lymph node

VCM

viable tumor cell condition medium

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Reagents
  8. Acknowledgements
  9. Conflict of interest
  10. References
  11. Supporting Information

Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.

FilenameFormatSizeDescription
eji2261-sup-0001-s1.pdf755KSupporting Information Figure 1.DC phenotype upon activation with tumor-cell supernatants.
eji2261-sup-0001-s1.pdf755KSupporting Information Figure 2. Phenotype of IL-2-expanded lymphocytes.
eji2261-sup-0001-s1.pdf755KSupporting Information Figure 3.CD39/CD69 expression by non-Treg.
eji2261-sup-0001-s1.pdf755KSupporting Information Figure 4. Purity of isolated CD4+CD25+Treg.
eji2261-sup-0001-s1.pdf755KSupporting Information Figure 5. Effect of IDO inhibition on ACM-induced suppression of cytotoxicity.
eji2261-sup-0001-s1.pdf755KSupporting Information Figure 6. Cytokine production by T cells in DC cocultures.
eji2261-sup-0001-s1.pdf755KSupporting Information Figure 7. Enrichment of CD39/CD69-expressing Treg in the CD4+CD8+ doublet population.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.