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.
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 []. These phagocytes program adaptive immunity by generating tumor-specific Treg cells, which are a major obstacle for antitumor immunity []. Treg cells are primed and activated mainly in the tumor-adjacent draining lymph nodes (TDLNs) by factors shed from the tumor []. Once primed, Treg cells travel to the tumor site, where they prevent effector T cells from eradicating the tumor []. 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 []. 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 [], or through tumor-released exosomes/microvesicles [], 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 [].
A prominent microenvironmental niche in tumors are dying tumor cells []. 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 []. 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 []. For instance, necrosis induces shedding of danger-associated molecular patterns, which activate TLRs on DCs []. 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 []. On the other hand, triggering of multiple immunosuppressive pathways upon priming with ACs has been recognized []. 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 []. However, cross-presentation of apoptotic cell (AC) derived antigens after chemotherapy does not necessarily culminate in antitumor immunity []. 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 []. 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 []. Understanding how priming by dying cells impacts antitumor immune responses might benefit cancer therapy.
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.
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).
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 [], and the lymphocyte activation marker CD69, expressed by Treg cells of cervical cancer patients [], 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.
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).
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 []. 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) [], 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) []. 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 []. 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.
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 []. 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 []. 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).
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 [], 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 [] as well as the adenosine receptor A2a inhibitor CSC [] 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.
A growing tumor or a tumor subjected to conventional therapy sheds tumor-derived factors/exosomes []. 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 [] 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 [].
Generation of suppressive Treg cells required activation of S1PR4 on DCs, likely due to S1P that is secreted by ACs []. S1P shifts LPS-induced maturation of DCs from Th1 to Th2 by suppressing IL-12 release and elevating IL-4 and IL-10 production []. 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 [].
The function of IL-27 in T-cell biology is ambiguous, varying between inflammatory (induction of Th1) and immunosuppressive []. IL-27 induces expression of the Th1 transcription factor T-bet [] and promotes CD8+ T-cell proliferation [] 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 []. 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 []. CD39 metabolizes ATP and ADP to AMP, the former being produced by activated T cells []. 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 []. Interestingly, ectonucleotidase expression and activity was increased in Treg cells of head and neck cancer patients []. 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 []. 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 []. Although TGF-β was connected with suppression of cytotoxic CD8+ T cells previously [], 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 []. 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 []. 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 []. 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
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 [] 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.
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) [] 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) [] 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) [] and respective isotype controls, were added to DC-T-cell cultures at day 2.
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.
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 []. Blocking of cytotoxic T cells was performed using CD8 antibody [] 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.
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 []. 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.
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).
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.
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).
Conflict of interest
The authors declare no financial or commercial conflict of interest.