The Heparan Sulfate Mimetic PG545 Modulates T Cell Responses and Prevents Delayed-Type Hypersensitivity

The heparan sulfate mimetic PG545 (pixatimod) is under evaluation as an inhibitor of angiogenesis and metastasis including in human clinical trials. We have examined the effects of PG545 on lymphocyte phenotypes and function. We report that PG545 treatment suppresses effector T cell activation and polarizes T cells away from Th17 and Th1 and toward Foxp3+ regulatory T cell subsets in vitro and in vivo. Mechanistically, PG545 inhibits Erk1/2 signaling, a pathway known to affect both T cell activation and subset polarization. Interestingly, these effects are also observed in heparanase-deficient T cells, indicating that PG545 has effects that are independent of its role in heparanase inhibition. Consistent with these findings, administration of PG545 in a Th1/Th17-dependent mouse model of a delayed-type hypersensitivity led to reduced footpad inflammation, reduced Th17 memory cells, and an increase in FoxP3+ Treg proliferation. PG545 also promoted Foxp3+ Treg induction by human T cells. Finally, we examined the effects of other heparan sulfate mimetics PI-88 and PG562 on lymphocyte polarization and found that these likewise induced Foxp3+ Treg in vitro but did not reduce Th17 numbers or improve delayed-type hypersensitivity in this model. Together, these data indicate that PG545 is a potent inhibitor of Th1/Th17 effector functions and inducer of FoxP3+ Treg. These findings may inform the adaptation of PG545 for clinical applications including in inflammatory pathologies associated with type IV hypersensitivity responses.

The first such inhibitor, PI-88, demonstrated promising results in mouse cancer models (15). However, a phase III clinical trial using PI-88 as an adjuvant in hepatocellular carcinoma therapy did not reach its primary end point (16).
PG545 is a member of a novel class of synthetic heparanase inhibitors, the PG500 family (17,18). A fully sulfated oligosaccharide attached to a lipophilic moiety, PG545 has improved pharmacokinetics and much weaker anticoagulant activity relative to other heparanase inhibitors, including PI-88 (19). PG545 is reported to function by blocking the catalytic center of heparinase and competing for the HSbinding domain (11,19). PG545 is currently under evaluation in human clinical trials for use in treating solid tumors after having demonstrated efficacy against tumor angiogenesis and metastasis in multiple animal studies (1,3,20,21). PG545 has also demonstrated efficacy against lymphoma and may also have utility in other, non-neoplastic inflammatory disorders, such as acute kidney injury, atherosclerosis, and viral infection (2,9,22,23). However, it remains unclear how PG545 impacts immune cells, particularly T-lymphocytes. This knowledge is likely to be important, given the key role of T-lymphocytes in cancer immunology and inflammation.
We sought to evaluate the impact of PG545 on T cells and T cell subsets, including pro-inflammatory Th1 and Th17 cells and anti-inflammatory FoxP3+ regulatory T cells (24,25). To this end, we studied this agent in the context of in vitro T cell activation and proliferation, antigenic responses in vivo, and in a skin hypersensitivity model.
We report that in vitro, PG545 promotes expansion of Treg while suppressing effector T cells, particular Th17 T cells. In vivo, PG545 suppresses antigen-specific T cell generation and cytokine production, leading to improvement in a Th17dependent delayed hypersensitivity model. Together, these data indicate that PG545 has profound effects on T cell polarization and function.

In vitro Suppression Assays
Suppression assays were performed as previously described (33). In brief, iTregs induced either in the absence or presence of PG545 were co-cultured with Cell Trace labeled responder CD4 cells and T cell-depleted splenocytes as antigen presenting cells. Activation was provided by 1 µg/ml of soluble anti-CD3 (145-2C11, Biolegend).

Ovalbumin Immunization
Naïve CD4 T cells from OT-II mice were stained with eFlour 450 cell proliferation dye (Thermo Fisher Scientific) and 1 × 10 6 of the cells adoptively transferred via intravenous tail injections into B6 recipients. The next day mice were immunized with 50 µg of OVA protein emulsified in 100 µL alum (Thermo Fisher Scientific).

In vivo Delayed Type Hypersensitivity Experiments
These experiments were performed as previously described (34). In brief, 8-10 week old mice were sensitized subcutaneously with 200 µg of mBSA (Sigma-Aldrich) emulsified in Complete Freund's Adjuvant (Santa Cruz Biotechnology) and challenged with 200 µg of mBSA solution in the foot pad of a hind limb. Control foot pad was injected with an equal volume of PBS. Footpad swelling was measured starting at day 0 using a digital caliper (Traceable). The reading of the "PBS" foot were subtracted from the "mBSA" foot for each individual mouse. PG545 dissolved in PBS was administered to mice intraperitoneally at a concentration of 400 µg/mouse at the indicated time point.

Generation of Murine Bone
Marrow-Derived Dendritic Cells BMDC were generated as described (28). Bone marrow from femurs of 6-10 week old mice was flushed out using a 27 G Precision Glide needle (BD Biosciences, Cat. No. 305109). Cells were plated at 1 × 10 6 cells in 10 ml of media per Petri Dish (Fisherbrand, Cat. No. FB0875711), supplemented with 10 ng/ml of recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) (STEMCELL Technologies, Cat. No. 78206.1) and 2 ng/ml of recombinant mouse IL-4 (Invitrogen, Cat. No. 14-8041-62). An equivalent amount of fresh media containing cytokines was added 3 days after plating, and 50% of media was changed on day 5 after plating. BMDCs were harvested for use on day 6.

Statistical Analysis
Statistical analysis was performed using Prism software, version 7.0 (GraphPad). Data are presented as mean SD. In samples with Gaussian distribution, an unpaired t-test was used to determine significant differences between groups or a 2-way ANOVA was used to identify effects of multiple parameters. A p < 0.05 was considered statistically significant.

PG545 Inhibits Effector T Cell Activation in vitro and in vivo
We first asked whether PG545 affects T cell activation in vitro.
To test this, we stimulated CD4+ T cells in a polyclonal manner. We observed that PG545 did not inhibit the activation of CD4+ T cells, as measured by the expression of CD69 and CD25 (Figures 1A,B).
We next examined cytokine production by these cells via intracellular staining. We observed that PG545 treatment resulted in altered cytokine production, including reduced IFNg and IL-17 in Th1 and Th17 polarizing conditions, respectively. This is seen for a representative staining example as well as across multiple samples (Figures 1C,D). This was also accompanied by decreased expression of T-bet and RORgt in the setting of PG545 treatment ( Figure 1G; Supplemental Figure 1A). PG545 also inhibited IL-17 production by activated total CD4 T cells, while sparing IFNg-competent T cells (Figures 1E,F), suggesting that PG545 acts early on during Th1 differentiation but not on mature Th1 memory cells.
Together, these data indicate that PG545 potently polarizes T helper differentiation away from Th1 and Th17 cells.
We next examined the impact of PG545 on adaptive immunity in vivo. To this end, we transferred Cell Tracelabeled, ovalbumin (OVA)-specific OT-II naïve CD4 cells from CD45.1 mice into naïve CD45.2 autologous recipient mice on day−2. These recipient animals then received either a single dose of PG545 or the same volume of phosphate buffered saline (PBS) as a control via intraperitoneal (i.p.) injection on day−1 Frontiers in Immunology | www.frontiersin.org followed by i.p. immunization with OVA on day 0. Then, on day 4 we euthanized the mice, isolated lymphatic tissue, and examined the proliferation and phenotype of these transferred cells via flow cytometry. A schematic of this protocol is shown in Figure 1H.
Following OVA immunization, transferred OT-II cells underwent rapid proliferation as measured by Cell Trace dilution. Mice that received either PG545 or control injections had equivalent proliferation of OT-II cells (Figures 1I,J). However, these cells from mice that received PG545 had lower expression of CD44 and PD-1 than control OT-II cells. This was seen for a representative staining example ( Figure 1K) as well as across samples from multiple mice (Figure 1L). Neither CD25 nor CD69 levels were altered (not shown). This is consistent with the effects of PG545 in vitro where we likewise did not observe effects on early T cell activation ( Figure 1A). Together, these data suggest that PG545 modestly impacts T cell activation but not proliferation.
We also examined PG545 effects on other aspects of murine health in this system. Mice treated with PG545 developed splenomegaly (Supplemental Figure 1B) a mild albeit not significant weight loss (Supplemental Figure 1C) without an increase in the lymphocyte count (Supplemental Figure 1D).
Together these data demonstrate that PG545 inhibits effector T cell phenotype without affecting their proliferation.

PG545 Enhances Induction of FoxP3+ Treg in vitro
Because CD44 expression is reported to influence the number of Foxp3+ Treg by promoting Treg homeostasis but inhibiting expansion (36,37) we considered whether PG545 might impact Treg. We did indeed observe that PG545 induced more Foxp3+ cells among OT-II T cells recovered from the spleen and draining lymph nodes of recipient mice. This was seen for a representative staining example (Figure 2A) as well as across samples from multiple mice ( Figure 2B). We believe that these cells represent de novo induced Foxp3+ cells rather than the expanded pre-existing Tregs, since the transferred naïve OT-II cells have undergone multiple rounds of division before expressing Foxp3 (Supplemental Figure 1F). In comparison to these effects with PG545, we did not observe an increase in Treg upon treatment with either another heparan sulfate mimetic PI88 ( Figure 2C) or with heparan sulfate itself using the same OT-II transfer model (data not shown). These data suggest that PG545 promotes the induction of Tregs in vivo. Given recent reports of the anti-inflammatory properties of PG545 in several systems (9, 22), we asked whether this agent promotes expansion of FoxP3+ Treg in vitro. To interrogate the effect of PG545 on T cells, we performed a Treg induction assay using a previously established protocol (33,38). Addition of PG545 increased the percentage of Foxp3+ Treg in the presence of IL-2 and TGFβ (Figures 2D,E). In addition, the total frequencies of Foxp3+ cell per sample were also increased in the presence of PG545 (Figure 2F). PG545 promoted Treg induction at a range of concentrations (Figures 2G,H). PG545 was more effective at promoting Foxp3+ Treg induction at lower TGFβ concentrations (Figures 2I,J) indicating a high sensitivity of T cells to PG545 during suboptimal Treg differentiation conditions.
To test whether PG545-induced Foxp3+ cells were functional, we used a previously described in vitro suppression assay that uses induced Foxp3+ Treg in conjunction with autologous, CFSE-labeled CD4+ T cells and autologous irradiated CD4-cells as antigen presenting cells (APC) (39). Using this system, we determined that Tregs induced in the presence of PG545 were equally potent in suppressing effector T cell activation in vitro (Supplemental Figure 1E).
We also tested the effects of PG545 on human cells. PG545 favored Treg induction of naïve human CD4 cells, most potently at low concentrations of TGFβ (Figures 2K,L).
Together, these data demonstrate that PG545 promotes the expansion of both human and mouse Treg numbers in vitro and in vivo. Moreover, these data suggest that iTregs derived in the context of PG545 are functionally competent.

PG545-Mediated Effects on Treg Induction Do Not Depend on HPSE Expression
We next asked whether the effect of PG545 on Treg induction was dependent on HPSE expression. To this end, we repeated our in vitro Treg induction assay using CD4+ T cells isolated from heparanase knock-out mice (HPSE −/− ) mice. To interrogate this, we bred HPSE −/− mice on a C57Bl6 background to strainmatched mice expressing a GFP/Foxp3 reporter (26,27).
We found that PG545 doubled Foxp3 frequencies of wild type as well as HPSE-deficient CD4 cells (Figures 3A,B). Interestingly, HPSE −/− CD4 T cells on their own produced higher frequencies of Foxp3+ cells, indicating that HPSE protein could inhibit iTreg induction. Nonetheless, the Treg-promoting effect of PG545 seems to be heparanase-dependent.

PG545 Inhibits Erk Signaling in Lymphoid Cells
We then asked how PG545 might promote Treg induction but inhibit Th1 and Th17 polarization. Erk1/2 signaling is known to inhibit Foxp3+ Treg induction and to promote Th1 polarization via suppression of Foxp3 and RORγt expression (40)(41)(42). We therefore asked whether PG545 might impact Erk1/2 signaling. To address this question, we examined Erk1/2 phosphorylation in CTLL2 T cells.
We observed a potent inhibition of basal Erk1/2 phosphorylation after treatment with PG545 (Figures 4A,B). Similarly, PG545 strongly diminished pErk signaling in primary activated mouse CD4 T cells (Figures 4C,D). These data suggest that PG545 inhibits basal Erk1/2 signaling. In agreement with previous findings (40,41), we observed a significant increase of Foxp3+ frequencies in the presence of Erk inhibitor (Figure 4E; Supplemental Figure 1G). This effect was dose-dependent, as higher concentrations of inhibitor suppresses Foxp3 induction and caused higher cell death (not shown). These findings are highly consistent with our observations that PG545 promotes Treg expansion and prevents polarization toward Th1.

PG545 Inhibits Th17 and Promotes Treg Cells in vivo
We next sought to evaluate the functional impact of PG545 in a model of Th1/Th17-dependent inflammation. One such model is the methylated bovine serum albumin (mBSA) induced delayed type hypersensitivity (DTH) model (34). Here, the sensitization phase is initiated by immunization with mBSA, which is delivered subcutaneously as an emulsion with complete Freund's adjuvant (CFA). The elicitation phase of the reaction is then triggered by a second injection of mBSA into the hind paws. Footpad inflammation is then measured 24 h later. To test the impact of PG545 in this system, mice were treated with PG545 via a series of i.p. injections. A schematic of this protocol is shown in Figure 5A.
We observed that mice treated with PG545 showed a dramatically blunted hypersensitivity response (Figures 5B,C). Of note, there was no difference in footpad swelling between mice that received a single injection of PG545 1 day post-sensitization vs. those that received an additional injection on the day of challenge (elicitation phase) ( Figure 5C).
To assess the effect of PG545 on priming vs. recall response, we treated DTH-immunized mice with PG545 at different time points. For this analysis, we also included HPSE −/− mice, previously used in the iTreg induction experiment (Figure 3A). Remarkably, irrespective of the treatment protocol, PG545 potently suppressed footpad swelling in all the experimental groups (Supplemental Figure 2A). While HPSE −/− mice mounted a normal DTH response, a single PG545 administration at day−6 fully prevented the swelling. Together these data demonstrate that PG545 has potent therapeutic effects in this model, irrespective of the administration.
We also assessed the impact of PG545 on the immune profile of these animals in this model. PG545 treated mice had a decrease in the draining LN cellularity (Figure 5D). This was accompanied with the decreased numbers of CD154+ CD4+ antigen-specific memory cells in the PG545-treated mice (Figures 5E,F). PG545 did not affect the systemic frequencies of Foxp3+ Tregs in this model (Figure 5G), but increased the proliferation of Tregs (Figure 5H).
Finally, we assessed the impact of PG545 on T cell polarization in this model. The fraction of IL-17 producing CD154+ T cells was dramatically diminished (Figures 5F,I). This also correlated with the cytokine staining obtained with the memory cell from the draining lymph node (Figure 1E). Likewise, memory cells from the mice treated at different times points all had impaired IL-17 production (Supplemental Figure 2B).
Since PG545 exhibits a strong inhibition of Th17 lineage, we wondered if this was the case for another Th17mediated autoimmune model experimental autoimmune encephalomyelitis (EAE). Surprisingly, PG545 administration was not able to make mice resistant to EAE, although it did significantly delay its onset ( Figure 5J). Interestingly, analysis of the spinal cord infiltrate revealed decreased frequencies of Th17 cells in mice treated with PG545, whereas Tregs remained unchanged (Figure 5K; Supplemental Figure 2C). This supports our observation in DTH model with respect to Th17 inhibition, but not the overall immune suppression.
Together these data indicate that PG545 is a potent inhibitor of DTH responses in mice and that PG545 may act in part by suppressing memory Th17 differentiation and priming.

PG545 Inhibits LPS Mediated Dendritic Cell Maturation
Considering the very potent anti-inflammatory effect of PG545 irrespective of the administration regimen, we wondered if PG545 could have an effect on the antigen presenting cells. For this, we administered PG545 into naïve mice and assessed their dendritic cell compartment 7 days later. After in vivo PG545 administration to naïve mice we observed lower relative DC frequencies in the spleen, probably due to splenomegaly (Figures 6A,B), but slightly higher frequencies in the lymph node relative to B220 lo CD3-cells. In contrast, DC frequencies relative to total live cells in both the spleen and lymphatic tissue were unchanged (Figures 6A-C). Next, we sought to analyze the DC response to LPS in the presence of PG545. For this, we generated bone marrow-derived dendritic cells and assessed their phenotype and functionality in vitro in the context of PG545. Strikingly, while the treatment with PG545 alone did not have a major effect of BMDC phenotype, PG545 greatly  diminished their LPS response as demonstrated by the decrease of maturation markers (Figure 6D). This is in contrast to the activation effect of PG545 on CpG treated BMDCs which was previously reported by Brennan et al. (23). Interestingly, we also observed a synergistic effect of PG545 and CpG toward DC maturation (data not shown). were analyzed using two-way ANOVA with Turkeys's multiple comparisons; (F,H)-two-tailed unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
In addition, while control BMDC potently activated T cells and promoted their proliferation in a polyclonal manner, PG545treated BMDC failed to do so (Figures 6E,F). Since the inhibition of BMDC could contribute to the reduced polarization toward Th17 phenotypes observed in our in vivo models, we also assessed the Th17 polarization in the presence of BMDC. We find that the fraction of Th17 cells after in vitro activation was reduced in the setting of PG545 (Figures 6G,H).

PI-88 and PG562 Favor Treg Induction in vitro but Do Not Suppress DTH
We also tested the effect of PI-88 and PG562 on Treg induction and T-cell mediated response. Both PI-88 and PG562 promoted Treg induction in vitro (Figures 7A-D) although less potently than PG545. This was true whether the Tregs were induced in the presence of anti-CD3/CD28 with TGFb (Figures 7A,B) or BMDCs with soluble anti-CD3 and TGFb (Figures 7C,D). However, only PG545 was able to prevent footpad inflammation in a DTH model when all three components were tested side by side ( Figure 7E). In agreement with the previous experiments, PG545 administration effectively suppressed the recall Th17 response in the draining LN of the animals, but neither PI-88 nor PG562 affected Th17 cells (Figures 7F,G). Of note, IFNg+ cells were not changed ( Figure 7H).
Together these data indicate that while all three heparinase inhibitors tested promote Treg induction in vitro, PG545 is unique in its ability to inhibit Th17-mediate DTH responses in mice.

DISCUSSION
Here we report that the HS mimetic PG545 affects T cell differentiation and function. In particular, PG545 selectively promotes the induction of anti-inflammatory Tregs while inhibiting the development of Th17 both in vitro and in vivo. Consistent with these effects, PG545 inhibits Erk1/2 signaling, a pathway known to inhibit Foxp3+ Treg induction and promote Th1 and Th17 polarization (40)(41)(42).
These data expand on our understanding of PG545 beyond its impact on tumor models, where it decreased angiogenesis and metastasis and improved survival (43)(44)(45)(46). More recently, several groups have shown a beneficial effect of PG545 treatment in atherosclerosis (47) and acute kidney injury (22). These effects were attributed to the reduction of pro-inflammatory markers, though neither increased Foxp3+ Treg nor reduced Th17 numbers were implicated.
Moreover, it appears that PG545 has both heparinase dependent and independent effects. Previously, the antiangiogeneic effects of PG545, for example, were attributed to inhibition of heparinase and heparan sulfate-binding angiogenic growth factors (17). However, our data indicate that PG545 promotes the expansion of Foxp3+ Treg independently of heparanase. Consistent with this, PG545 was recently reported to have apoptotic effects on lymphoma cell lines that lack heparanase expression (2).
One question that this study raises is how an antiinflammatory effect of PG545 observed here reconciles with its anti-tumor activity shown in other studies. Since cytotoxic T cells are the main effector T cells involved in cancer immunotherapy, it may be that these are less sensitive to the effect of PG545 treatment than tumor cells (48). It may also be that the effects of PG545 are context dependent. A recent study has shown that MEK inhibition can reactivate tumorinfiltrating CD8 lymphocytes by preventing their exhaustion (48). Finally, it may also be that the impact of PG545 on Erk signaling or other pathways in those models may trump effects on lymphocytes.
It is tempting to consider that both the anti-tumor and anti-inflammatory properties of PG545 may be attributable in part to inhibition of Erk1/2 signaling. However, PG545 has effects that are difficult to entirely attribute to Erk1/2 inhibition. For example, PG545 is reported to decrease glucose uptake, downregulate glycolytic machinery, inhibit Wnt signaling, and induces autophagy in cancer cell lines (2,43,44). Moreover, while Erk1/2 signaling inhibits Th17 polarization in some settings, it promotes Th17 in others, suggesting that both the regulation of these signaling cascades and their inhibition is likely to be complex (40,(49)(50)(51). Finally, we demonstrate effects similar to Erk signaling on Foxp3+ Treg but do not conclusively demonstrate that these are Erk-mediated. In the aggregate, our interpretation of the data is that it suggests that PG545 may acts via multiple mechanisms of which Erk1/2 inhibition is a part.
A report from Brennan et al. (23) suggested an activation effect of PG545 on dendritic cells. However, while Brennan et al. activated BMDC with the CpG mimic we used LPS stimulation, indicating the context-dependent properties of the HS mimetic.
Overall, we show that PG545 promotes regulatory T cells in vitro. This effect is accompanied with the inhibition of Th17 cells and to some extent Th1 cells. PG545 acts on T cells in heparanase-independent manner and impairs Erk signaling in proliferating cells without affecting an early activation program. PG545 administration effectively suppressed DTH in mice. These findings may inform the adaptation of PG545 for clinical applications including in inflammatory pathologies associated with DTH.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

ETHICS STATEMENT
All experiments were approved by the Stanford IACUC.

ACKNOWLEDGMENTS
We would like to thank K. Dredge (Paragen Bio) and E. Hammond (Zucero Therapeutics) for providing HS mimetics and discussing the data, Peggy Ho (Steinman lab, Stanford University) for help with the CFA emulsification. We thank Stanford Shared FACS facility for their assistance.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.00132/full#supplementary-material FACS profile of the CD4 T cell cytokine staining from DTH mice with various PG545 treatment. (C) Representative bar diagram with Foxp3+ and IL-17+ cell frequencies among spinal cord CD3+CD4+ T cells in EAE mice. Data shown are for mean ± SD using a two-tailed unpaired t-test or two-way ANOVA test. * p < 0.05, * * p < 0.01.