Human Placental Pericytes Poorly Stimulate and Actively Regulate Allogeneic CD4 T Cell Responses

PC Express an Immunophenotype Distinct From That of EC

We compared placental PC and autologous human umbilical vein EC for expression of immunologically significant surface molecules under basal and cytokine-stimulated conditions. Like EC, PC expressed MHC class I molecules but not class II molecules under basal culture conditions; both class I and class II MHC molecule expression were induced following treatment with IFN-γ (Table). However, MHC molecule expression was lower on PC than EC. Neither PC nor EC expressed the costimulatory molecules CD80 or CD86, but both expressed positive costimulatory molecules CD58, CD40, CD275, CD137L, and CD252, although PC expression levels were somewhat lower. PC lacked glucocorticoid-induced TNFR-related protein (GITR) ligand, an inhibitor of regulatory T cell (Treg) function, which was expressed basally on EC and increased by treatment with IFN-γ or tumor necrosis factor. PC more highly expressed the inhibitory molecules CD274 (programmed death [PD]-L1) and CD273 (PD-L2) than did EC, especially following treatment with IFN-γ. Unlike EC, PC did not express the adhesion molecules CD106 (vascular cell adhesion molecule-1) or E-selectin, but they did express similar levels of CD54 (intercellular adhesion molecule-1), which was further induced on both cell types following cytokine treatment (Table). Overall, compared with umbilical vein EC, human placental PC displayed an immunophenotype expected to be less likely to activate T cell responses.

PC⁺ Do Not Effectively Stimulate Allogeneic CD4 T Cells

We directly compared the ability of placental PC and umbilical vein EC to induce allogeneic CD4 T cell responses in vitro. Peripheral blood CD4 T cells were cultured with allogeneic IFN-γ pretreated (MHC class II molecule–expressing) vascular cells (designated PC⁺ or EC⁺), as well as with untreated (MHC class II molecule–negative) cells (designated PC⁻ and EC⁻). CD4 T cells cocultured with allogeneic PC⁺ produced very little IL-2 or IFN-γ compared with cocultures with EC⁺ (Figure 1A). Neither cytokine was detected in cocultures of CD4 T cells with allogeneic PC⁻ or EC⁻. A similar percentage of CD4 T cells cocultured with PC⁺ or EC⁺ expressed the activation markers CD69 at 24 hours and CD25 at 72 hours (Figure 1B), which were absent on freshly isolated CD4 T cells or CD4 T cells cocultured with PC⁻ or EC⁻. A greater percentage of CD4 T cells cocultured with EC⁺ than with PC⁺ were in cell cycle or were dying at day 3 assessed by propidium iodide staining (Figure 1C). In contrast to CD4 T cells cocultured with allogeneic EC⁺, CD4 T cells cocultured with allogeneic PC⁺ did not proliferate after 7 days, as assessed by carboxyfluorescein succinimidyl ester dilution (Figure 1D) or 5-bromo-2′-deoxyuridine incorporation (data not shown). The limited ability of PC⁺ compared with EC⁺ to activate allogeneic CD4 T cells was a consistent feature of multiple donor combinations (Figure 1E).

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Figure 1.

PC⁺, unlike EC⁺, do not stimulate resting allogeneic CD4 T cell responses. PC⁻, PC⁺, EC⁻, and EC⁺ were cocultured with allogeneic CFSE-labeled CD4 T cells. A, Medium was analyzed for the presence of IL-2 and IFN-γ by ELISA after 24 hours. B, T cells were analyzed by flow cytometry for expression of CD69 at 24 hours and CD25 at 72 hours. C, Propidium iodide (PI) staining of cocultured CD4 T cells after 72 hours. D, CD4 T cell proliferation assessed after 7 days of coculture by CFSE dilution. E, Pooled data demonstrating CD4 T cell proliferation after 7 days of coculture. Shown are representative results from 1 of 4 experiments in A, C, and D and pooled data from 5 independent experiments in B and E; **P<0.01; ANOVA/Bonferroni post test.

The induction of CD69 and CD25 suggests that CD4 T cells do recognize class II MHC molecules on PC⁺ and therefore might be able to restimulate activated T cells, which have minimal need for positive costimulation. We generated activated T cells by culturing CFSE-labeled CD4 T cells with allogeneic EC⁺ for 7 days and collecting CFSE^low CD4 T cells by cell sorting. Activated CD4 T cells proliferated in response to coculture with PC⁺ autologous to the primary stimulation EC⁺ but not to unrelated (“third-party”) PC⁺, assessed by 5-bromo-2′-deoxyuridine labeling (Figure 2A). However, secondary proliferation to autologous PC⁺ was less than the restimulation response to autologous EC⁺ (Figure 2A). Such differences were consistent over multiple donors (Figure 2B).

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Figure 2.

PC⁺ stimulate preactivated allogeneic CD4 T cells, but less well than EC⁺. CFSE-labeled CD4 T cells were cultured with EC⁺ for 7 days. CFSE^low CD4 cells were collected by cell sorting, rested, and then restimulated by PC⁺ autologous to EC⁺ used for priming or by third-party PC⁺. Restimulation by EC⁺ served as a positive control. A, Activated CD4 T cell proliferation determined by 5-bromo-2′-deoxyuridine (BrdU) incorporation over 4 days of restimulation. B, Data were normalized by referencing to the positive control and then pooled for assessing statistical significance. Shown are representative results from 1 of 4 independent experiments in A and pooled data from 4 independent experiments in B; *P<0.05; t test.

To directly assess whether the difference between PC⁺ and EC⁺ arises from differences in costimulation, we compared cocultures of vascular cells and CD4 T cells stimulated by the polyclonal-activating lectin phytohemagglutinin-L (PHA-L), an assay that is independent of MHC class II molecules but relies on costimulation provided by accessory cells. PC⁻ or PC⁺ required increased concentrations of PHA to stimulate maximal cytokine production and proliferation by CD4 T cells compared with EC⁻ or EC⁺ (Figure 3A). Furthermore, the maximum level of these responses was less in cocultures with PC than EC at saturating concentrations of PHA. As expected, no difference in CD25 induction was observed, because this response is largely mediated by T cell receptor engagement by PHA. We specifically assessed the role of negative costimulation provided by PD-1 ligands by means of antibody blocking, again using PHA to amplify the response. Addition of neutralizing antibodies to PD-1 ligands increased CD4 T cell proliferation to PC⁺, but proliferation was still less than that observed in parallel EC⁺ cocultures at the same dose of PHA (Figure 3B). Thus, engagement of PD-1 contributes to, but does not fully explain, the poor accessory functions of PC. We conclude that the net balance of costimulation provided by PC is less than that provided by EC, likely resulting from quantitative differences in multiple different costimulators.

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Figure 3.

PC support PHA-induced T cell mitogenesis less well than EC, due in part to expression of inhibitory PD-1 ligands. A, CFSE-labeled CD4 T cells were cocultured with allogeneic stimulator cells plus varying concentrations of the polyclonally activating lectin PHA. IL-2 and IFN-γ levels were measured after 24 hours by ELISA. Activation was assessed by CD25 expression, and proliferation was assessed by CFSE dilution after 48 hours. B, Proliferation of CFSE-labeled CD4 T cells cocultured with allogeneic stimulator cells in the presence of 0.5 μg/mL PHA and the indicated neutralizing antibodies for 72 hours. Shown in A are representative results from 1 of 3 experiments. B, Representative results from 1 of 3 experiments; error bars indicate SEM between triplicate wells within 1 experiment.

PC⁺ Induce CD4 T Cell Anergy

Because PC⁺ induced expression of the activation markers CD69 and CD25 on CD4 T cells, we investigated whether T cells demonstrate any functional change as a consequence of coculture with PC⁺. CD4 T cells were briefly cultured with PC⁺ for 24 hours and then restimulated with either EC⁺ from the same donor as the PC⁺ or third-party EC⁺. In these secondary cocultures, the response to matched EC⁺ resulted in less IL-2, IFN-γ, and T cell proliferation compared with secondary cocultures restimulated by third-party EC⁺ (Figure 4A and 4B). This functional alteration depends on recognition of class II molecules on PC because no inhibition was seen in primary cultures with PC⁻ instead of PC⁺, and inclusion of a human MHC class II (human leukocyte antigen-DR) blocking antibody in the primary coculture significantly decreased the degree of secondary inhibition (Figure 4C). In contrast, blocking PD-1 ligands in the primary culture had little effect (not shown).

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Figure 4.

CD4 T cells cultured with PC⁺ become desensitized to secondary allogeneic stimulation. Allogeneic CFSE-labeled CD4 T cells were cocultured with PC⁺ for 24 hours then restimulated by EC⁺ autologous to the primary-stimulation PC⁺ (matched EC⁺) or by third-party EC⁺. A, Media were collected after 24 hours of restimulation and analyzed for the presence of IL-2 and IFN-γ by ELISA. B, CD4 T cell proliferation after 7 days of restimulation, normalized to secondary proliferation responses of T cells cultured initially with EC⁺. C, The enhanced suppression to matched EC⁺ in secondary cultures was largely reversed by inclusion of an antibody blocking HLA-DR in the primary PC⁺ coculture. D, Activated CD4 T cells cocultured with PC⁺ expressed increased GRAIL, ITCH, and CBL-B transcripts and decreased FoxP3 transcripts compared with T cells cocultured with EC⁺. Shown are representative results from 1 of 8 experiments in A and B, 1 of 2 experiments in D, and pooled data from 4 experiments in C; *P<0.05, **P<0.01; Mann-Whitney test (B); t test (C and D).

Recognition of HLA-DR on PC⁺ could induce a degree of alloantigen-specific clonal anergy in CD4 T cells or could induce alloantigen-specific Treg cells capable of inhibiting responses in secondary cultures. We assessed Treg induction by immunophenotyping and by functional suppression assays, finding that coculture with PC⁺ neither induced T cell expression of characteristic Treg markers (CD4⁺CD25⁺CD127^lowFoxp3⁺) nor rendered T cells capable of suppressing alloresponses of freshly isolated T cells to EC⁺ (data not shown). We also saw no evidence for the Treg-induced cytokines IL-10 or TGF-β or any effect of neutralizing these inhibitory cytokines (data not shown). We next examined CD4 T cell expression of several genes associated with human T cell anergy, specifically GRAIL, ITCH, and CBL-B.^26,27 Activated CD25+ CD4 T cells cocultured with PC⁺ for 72 hours expressed increased levels of GRAIL, ITCH, and CBL-B mRNA and decreased levels of Foxp3 mRNA compared with T cells cocultured with EC⁺ (Figure 4D). These experiments suggest that recognition of class II MHC molecules on allogeneic PC⁺ renders CD4 T cells clonally anergic.

PC⁺ Inhibit CD4 T Cell Proliferation Across a Semi-Permeable Membrane

Because many stromal cell populations reportedly modulate T cell responses via the secretion of diffusible inhibitory factors, we investigated whether PC⁻ or PC⁺ could affect T cell responses independent of cell contact. Freshly collected PC⁺ conditioned medium showed a small inhibitory effect on CD4 T cell proliferation to EC⁺, but the effect was inconsistent and the factor(s) appeared unstable (data not shown). In contrast, PC⁻ or PC⁺, but not EC⁻ or EC⁺, cultured across semi-permeable membranes from allogeneic EC⁺/CD4 T cell cocultures, consistently inhibited CD4 T cell proliferation (Figure 5A), and PC⁺ were more potent than PC⁻. Inhibition was similarly observed when CD4 T cells were cocultured with EC⁺ and the superantigen toxic shock syndrome toxin-1 in the presence of PC⁻ or PC⁺ across semi-permeable membranes (data not shown).

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Figure 5.

PC inhibit CD4 T cell proliferation across a semi-permeable membrane, an effect partially mediated by IL-10 and TGF-β but not mediated by depletion of l-tryptophan or l-arginine or by production of nitric oxide or PGE2. CFSE-labeled CD4 T cells were cocultured with allogeneic EC⁺ in the presence of EC, PC⁻, or PC⁺ across a semi-permeable membrane (labeled [EC], [PC], and [PC⁺]) and various other molecules as indicated. A to F, T cell proliferation was assessed by CFSE dilution after 7 days. Parallel CD4/EC⁺ cocultures carried out in the presence of EC⁺ across a semi-permeable membrane served as a positive control used to generate normalized proliferation values for comparison across experiments using cells from different donors. Shown are pooled data from 8 experiments in A and 4 experiments in F; *P<0.05; ANOVA/Bonferroni post test.

We examined several mechanisms used by other stromal cell types to inhibit T cell responses. We were particularly interested in those mediators that were IFN-γ inducible in PC, because PC⁺ demonstrated greater inhibition. IFN-g treatment of PC increases mRNA levels of the tryptophan metabolizing protein IDO (Supplemental Figure Ia), an enzyme known to be involved in human smooth muscle cell and dendritic cell inhibition of T cell responses via depletion of the essential amino acid l-tryptophan.^23,28,29 However, PC⁺ expression of IDO was not greater than IDO expression by donor-matched EC⁺ (Supplemental Figure Ib), which do not suppress T cell proliferation, and neither addition of exogenous tryptophan nor addition of the IDO-antagonist 1-methyltryptophan reverses the inhibitory effect of PC⁺ in cocultures (Figure 5B). We also investigated whether PC might deplete cocultures of l-arginine, as human embryonic stem cells may suppress T cell responses by arginase-1–mediated l-arginine depletion.³⁰ Arginase-1 was not detected in cell lysates from either PC⁻ or PC⁺, but arginase-2 and the arginine uptake membrane transporter CAT2B were expressed and increased in PC⁺ compared with PC⁻ (Supplemental Figure II). However, the addition of exogenous l-arginine did not abrogate the inhibitory effect of PC⁺ in semi-permeable membrane assays (Figure 5C). We also examined PC production of several mediators known to suppress T cell proliferation, namely nitric oxide (NO), PGE2, TGF-β, and IL-10. Whereas inducible nitric oxide synthase (iNOS) mRNA levels were increased in PC⁺ versus PC⁻, iNOS protein was not detected by immunoblotting, and increased NO was not detected in the media of PC⁻ or PC⁺ cultures (Supplemental Figure IIIa to IIIc). Furthermore, treatment of semi-permeable membrane cocultures with the specific iNOS inhibitor 1400W failed to reverse the inhibition (Figure 5D). PGE2 synthesis from arachidonic acid occurs constitutively by the action of COX-1 and inducibly through COX-2. COX-2 expression was slightly increased in PC⁺ versus PC⁻ (data not shown), and PC⁺ produced increased levels of PGE2 compared with PC⁻ (Supplemental Figure IVa). However, medium from EC⁺/CD4 T cell cocultures with PC⁻ or PC⁺ across a semi-permeable membrane contained less PGE2 at 24 hours than medium from cocultures with EC across a semi-permeable membrane (Supplemental Figure IVb). Furthermore, suppression of CD4 T cell proliferation was not reversed by treatment of cocultures with the cyclooxygenase inhibitor indomethacin (Figure 5E). Although neither TGF-β nor IL-10 appeared to be induced in PC by IFN-γ treatment, addition of neutralizing antibodies to IL-10, TGF-β, or both significantly relieved the inhibition of CD4 T cell proliferation caused by PC⁺ across a semi-permeable membrane (Figure 5F). However, the effect of neutralizing both cytokines was neither additive nor complete, suggesting that although IL-10 and TGF-β contribute to the inhibitory milieu, perhaps acting through the same pathway, other mechanisms must contribute as well.

We examined whether the presence of PC⁺ across a semi-permeable membrane renders T cells less responsive to restimulation, as was observed in cocultures with cell-cell contact. T cells recovered from PC⁻ and PC⁺ semi-permeable membrane cocultures responded similarly to control (EC semi-permeable membrane) T cells in secondary stimulation assays to EC⁺ (Supplemental Figure V). In other words, PC⁺ inhibited T cell responses across a semi-permeable membrane but direct contact was necessary to induce clonal anergy.

Finally, we addressed whether placental PC are unique in their immunologic properties by investigating the immunogenicity of PC isolated from another tissue source. Human brain vascular PC were assessed for MHC, costimulatory, and adhesion molecule expression under basal and IFN-γ stimulated conditions (Supplemental Table I). Brain PC expressed a relatively similar immunophenotype as compared with placental PC except that not all brain PC expressed class II MHC molecules following IFN-γ treatment. Like T cells cocultured with placental PC⁺, CD4 T cells cocultured with brain PC⁺ did not produce IL-2 (Supplemental Figure VIa) or proliferate (Supplemental Figure VIc), despite induced CD25 expression (Supplemental Figure VIb). Brain PC also inhibited alloreactive CD4 T cell proliferation to EC⁺ across a semi-permeable membrane (Supplemental Figure VId). Although these observations do not permit generalization to all PC, they do suggest that placental PC are not unique in their immunomodulatory functions.