Acid-driven immune suppression by pHLIP-fused PD-L1 under inflammatory conditions

doi:10.21203/rs.3.rs-2438827/v1

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Acid-driven immune suppression by pHLIP-fused PD-L1 under inflammatory conditions

https://doi.org/10.21203/rs.3.rs-2438827/v1

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Programmed cell death–ligand 1 (PD-L1)/PD-1 axis is crucial for maintenance of immune homeostasis and its impairment partially accounts for the pathogenesis of inflammatory diseases. Hence, augmenting PD-L1/PD-1 signals represents a novel strategy to prevent destructive inflammation and induce immune tolerance. Recently, we developed a new cargo by conjugating the ectodomain of PD-L1 with pHLIP, a low pH-responding and membrane-inserting peptide, and demonstrated its potent immune-suppressive activity under acidic conditions in vitro. Herein, we further showed that PD-L1-pHLIP well responded to relatively high acidic buffer, while it could not inhibit T cell expansion in weakly acidic solutions. Furthermore, in a mouse model of acute intestinal inflammation, PD-L1-pHLIP treatment prolonged survival time and attenuated colitis in mice subjected to 6% dextran sulfate sodium (DSS) instead of 3% DSS. The different efficacy was due to the distinct acidity in the lesions, which facilitated PD-L1-pHLIP accumulation in the niche of 6% DSS-triggered severe inflammation. Mechanistic investigations revealed that PD-L1-pHLIP inhibited the release of proinflammatory cytokines in infiltrating macrophages and other immune cells in a PD-1-dependent and/or –independent fashion. Taken together, this study highlights PD-L1-pHLIP as a novel therapeutic avenue for inflammatory diseases.

Biological sciences/Immunology/Immunotherapy

Biological sciences/Drug discovery

PD-L1

PD-1

pHLIPs

DSS

PD-L1, a ligand for (PD)-1, is expressed by CD4⁺ and CD8⁺ T cells, B cells, dendritic cells, macrophages, and nonhemopoietic tissues^1,2. The interaction between PD-L1 and PD-1 inhibits T cell activation and plays an important role in maintenance of immune homeostasis and resolution of inflammation^3,4. There is mounting evidence that the impairment of PD-L1/PD-1 signals contributes largely to the pathogenesis of autoimmunity and inflammatory diseases, making it rational to develop PD-1 agonists as therapeutics against unresolved inflammation ^5,6.

pH (Low) Insertion Peptides (pHLIPs) are a family of soluble ཞ36 amino acid peptides, which is monomeric and largely unstructured at neutral and high pH⁷. If the environment is acidic, pHLIPs fold and insert across the membrane to form a stable transmembrane helix, thus preferentially locating itself in acidic diseased tissues^8,9. Recently, we have developed a new cargo by engaging the ectodomain of murine PD-L1 to the N terminus of pHLIPs, and demonstrated its potent immune-suppressive activity under acidic conditions in vitro¹⁰. Given that acidosis is the hallmark of microenvironment of acute inflammation attributed to augmented glycolysis and lactate efflux in infiltrating immune cells ^11–13, we assume that PD-L1-pHLIP may respond to the drop of pH and accumulate in inflamed lesions, and thereby deliver negative signals to PD-1-expressing immune cells, ultimately dampen inflammatory responses. Of note, the pH₅₀ of membrane insertion of pHLIPs utilized in this study is approximately 6.1 ^14–16, rendering the possibility that PD-L1-pHLIP is unable to anchor on the cellular membrane in the niche with relatively high pH and thereby loses its immune-suppressive function. To address this issue, the function of PD-L1-pHLIP was evaluated in pH6.8 and 6.1 buffer respectively in this report. Furthermore, two doses of DSS (3% and 6%) was administrated to establish the model of intestinal inflammation and the efficacy of PD-L1-pHLIP was determined.

Mouse strains, colitis model and PD-L1-pHLIP treatment

Female, 6–8 week-old C57BL/6J and Balb/c mice were obtained from Charles River and OT-II mice were purchased from Cyagen Biosciences. Animals were handled according to protocols approved by the institutional committee for animal use. Mice received sterile water with 3% or 6% DSS (MW 36,000–50,000; MP Biochemicals) for 5–7 days. PD-L1-pHLIP or PD-L1-Fc, prepared as described previously¹⁰, were injected intraperitoneally at the dose of 5mg/kg every other day following the beginning of DSS instillation.

Cell And Organ Culture Preparation

The splenocytes of BALB/c mice were isolated by gradient centrifugation and stimulated with Dynabeads™ Mouse T-Activator CD3/CD28(ThermoFisher, 11452D) for 72h in the indicated pH buffer titrated by lactic acid or hydrochloric acid. PD-L1-pHLIP or HER2-pHLIP (human HER2 ectodomain fused with pHLIP, prepared by our lab) was added at titrated concentrations. RAW264.7 cell line (purchased from ATCC) were stimulated with 1µg/ml LPS (Sigma-Aldrich, L4516) for 24h. In some settings, neutralizing mAbs to murine PD-1 or IgG2a isotypes (10 µg/ml, Biolegend) were added. The supernatant was collected and stored at -20°C until analyzed.

Colons were washed in ice-cold PBS supplemented with 100 U/ml penicillin/streptomycin, cut in 0.5-cm pieces and cultured in 24-well-flat-bottom plates (Corning, 3524) in serum-free RPMI-1640 medium for 24h. The supernatant was harvested and stored at -20°C until analyzed.

Cfse Lymphocyte Proliferation Assay

Splenocytes were stained with 5 µM CFSE (ThermoFisher) in PBS for 10 min at 37℃and then cells were washed two times with RPMI-1640 medium and resuspended at a concentration of 2.5 × 10⁶ cells/ml. The CFSE-stained cells were transferred to 96-well plates (200 µl/well) and stimulated with Dynabeads™ Mouse T-Activator CD3/CD28 for 72h in the indicated pH buffer. PD-L1-pHLIP or HER2-pHLIP (0.1 µg/ml) was added. The cells were harvested and stained with APC-anti-mouse-CD8 and FITC-anti-mouse-CD4 (Biolegend) at 4°C for 30 min and subsequently used for flow cytometry analyses.

Brdu Cell Proliferation Assay

BrdU Cell Proliferation Assay Kit (CST, 6813S) was used to detect lymphocyte proliferation according to the manufacturer’s protocols.

Cytokine Detection By Elisa

IFN-γ, IL-2, IL-22, IL-17A, IL-1β, TNF-α, IL-6 levels in the supernatants were determined by commercial ELISA assay kits (Biolegend).

Organ Imaging

pHLIP or PD-L1-pHLIP were conjugated with Cy5.5 dye according to the manufacturer’s protocols (Abcam, USA). Cy5.5-labeled pHLIP or PD-L1-pHLIP was injected intraperitoneally at the dose of 20µg/mouse. 24h later, mice were visualized by bioluminescence imaging (PerkinElmer, USA).

Flow Cytometry

Splenic lymphocyte were stimulated with Dynabeads™ Mouse T-Activator CD3/CD28 and incubated with PE-labeled PD-L1-pHLIP at 0.1µg/ml for 72h in pH6.8 or 6.1 buffer. Single cell suspensions (1×10⁶) were stained on ice for 30 min with antibodies in FACS buffer (PBS, 1% BSA, and 1 mM EDTA). The antibodies used were following: FITC-conjugated NK1.1, CD19, CD11b, CD11c, CD25, CD69, PE-conjugated CD4, CD8, PD-1, APC-conjugated CD3, F4/80. All antibodies were purchased from Biolegend. Flow cytometry was performed on a FACSAria II (BD Biosciences) and data analysis was performed by using the FlowJo software.

Measurement Of Lactic Acid Production And Ph

Colons were homogenized and extracted with a tissues/Ethanol ratio of 1:8 (1 h at 4°C) followed by centrifugation at 10,000g. Lactic acid contents were determined by L-lactate assay kit I (Eton Bioscience, 1200012002). pH in the colon homogenates were measured by electronic pH meter (sartorius, PB-10).

Histological Analysis, Immunohistochemistry And Immunofluorescence

Colon tissues were isolated, washed in PBS and either fixed with 10% formaldehyde (Xilong scientific, 200-001-8) or embedded in OCT(Servicebio, G6059) compound. Paraffin-embedded sections were stained for hematoxylin-eosin (HE). Colitis score was undertaken according to the criteria described previously³⁹. For immunohistochemistry of mouse colon tissue, slides were incubated with primary antibody against SLC26A3 (1:200) (Novusbio, NBP1-84450) in PBS containing 1% BSA and 10% goat serum. Biotinylated secondary antibody (Dako) were added and incubated at room temperature for 1 h. Streptavidin-HRP (BD Pharmingen) was added, and after 40 min the sections were stained with DAB substrate and counterstained with hematoxylin. For immunofluorescence staining, snap frozen slides were stained with antibodies against CD206, CK19, CD11c, CD45 or Ly6G (DAKO) followed by secondary donkey antibodies labeled with AlexaFlour 488, counterstained with DAPI and analyzed by immunofluorescence. Images were acquired by image processing software CaseViewer on upright light/fluorescent Imager A2 microscope (Zeiss) equipped with an AxioCam camera or on a confocal microscope (Leica SPE).

Statistical analysis

Data are expressed as the mean ± SEM, and the differences between groups were analysed using either unpaired two-tailed Student’s t test or one-way ANOVA with Bonferroni correction. A value of p < 0.05 was considered statistically significant.

PD-L1-pHLIP inhibit T effector function in pH6.1 rather than pH6.8 buffer

Firstly, we determine the inhibitory effects of PD-L1-pHLIP on T cell responses upon TCR stimulation under weakly acidic (pH6.8) and highly acidic (pH6.1) conditions respectively. In consistent with our previous study¹⁰, soluble PD-L1-pHLIP did not exhibit the suppressive function on lymphocyte proliferation in neutral aqueous solution, whereas actively inhibited lymphocyte expansion in pH6.1 buffer titrated by lactic acid (Fig. 1a). Intriguingly, PD-L1-pHLIP lost its inhibitory function on anti-CD3/CD28-stimulated polyclonal proliferation of lymphocyte in pH6.8 buffer (Fig. 1A). To accurately clarify the inhibitory effects of PD-L1-pHLIP on T cell subsets, carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution was performed. PD-L1-pHLIP, indeed, significantly suppressed CD4⁺ and CD8⁺T cell expansion in pH6.1 buffer (Fig. 1B). However, the inhibitory effects were not observed in pH6.8 solutions (Fig. 1b). In accord with this, PD-L1-pHLIP decreased IFN-γ and IL-2 secretion by lymphocytes in pH6.1 buffer instead of pH6.8 solutions (Fig. 1c). Furthermore, the effects of PD-L1-pHLIP on inhibition of lymphocyte proliferation and proinflammatory cytokine production was dose-dependent (Fig. 1d ,1e). Next, we detected the effects of PD-L1-pHLIP on the expression of T cell activation biomarkers (e.g. CD25 and CD69). Under acidic conditions, TCR stimulation enable increased expression of CD25 and CD69 on CD4⁺T cells and PD-L1-pHLIP treatment reduced their expression levels in pH6.1 rather than pH6.8 buffer (Fig. 1f). We also evaluated the inhibitory function of PD-L1-pHLIP in hydrochloric acid-titrated pH6.1 and 6.8 buffer. Similar results were seen (Fig.S1). To determine the function of PD-L1-pHLIP on antigen-specific T cell expansion, CD4⁺T cells were isolated from OTⅡ-transgenic mice and pulsed with OVA_323-339. Lactic acid treatment had no significant influence on CD4⁺T cell proliferation. Importantly, PD-L1-pHLIP dramatically reduced OVA-pulsed T cell expansion in pH6.1 buffer but not in pH6.8 solutions (Fig. 1g). Therefore, we make a conclusion that PD-L1-pHLIP displays its immuno-suppressive function under highly acidic conditions, which is lost under weakly acidic conditions. This may be attributed to the fact that in weakly acidic buffer PD-L1-pHLIP is incapable of inserting into the cellular membrane and thereby does not crosslink to its ligand PD-1, which is prerequisite for initiation of PD-L1/PD-1 signals^17,18 .

Pd-l1-phlip Displays On The Surface Of Several Types Of Immune Cells Under Acidic Conditions

Our previous study has shown that PD-L1-pHLIP had ability to span across the cellular membrane by conformation changes of pHLIP in response to low pH and retained on the surface of T and B cell lines and primary mouse and human lymphocytes¹⁰. Herein, we further determine cell distribution of PD-L1-pHLIP in pH6.8 and 6.1 buffer. In agreement with previous study¹⁰, no fluorescence was visible when PE-labeled PD-L1-pHLIP was incubated with anti-CD3/CD28-stimulated lymphocytes in neutral aqueous solutions (data not shown). Unexpectedly, in pH6.8 buffer, fluorescence-labeled PD-L1-pHLIP was easily seen on the surface of several types of lymphocytes including NK, dendritic cell, monocyte, macrophage and B cells (Fig. 2, upper panel). The magnitude of fluorescence was highest on B subset (Fig. 2, upper panel). Interestingly, the fluorescence on T lymphocyte was absent, indicating that PD-L1-pHLIP could not insert into cellular membrane of this subset under acidic conditions, which was further supported by the inability of the insertion of PD-L1-pHLIP in pH6.1 buffer (Fig. 2, upper panel). Notably, the intensity of fluorescence on immune cells detected by flow cytometry was significantly lower in pH6.8 buffer compared with that in pH6.1 solutions (Fig. 2, lower panel). This may account for distinct effects of PD-L1-pHLIP in pH6.8 and 6.1 buffer as described above. The relatively low magnitude of PD-L1-pHLIP on the cell surface in pH6.8 buffer is not sufficient to deliver negative signals to T cells and inhibit effector function.

Low Ph And Lactate Accumulation In The Lesions Of Dss-induced Colitis

To validate the therapeutic potentials of PD-L1-pHLIP in vivo, two doses of DSS (3% and 6% DSS in water) were administrated to establish murine model of acute intestinal inflammation and the acidity in the diseased colonic tissues were detected. On day 3 post the initiation of DSS instillation, pH value in the lesions decreased slightly compared with naïve colons (Fig. 3a). On day 7, pH significantly dropped at ཞ0.4 unit compared with the counterparts collected on day 3, regardless of 3% or 6% DSS-instilled mice. Of importance, 6% DSS instillation resulted in lower pH and more acidic in the diseased tissues than that in mice drinking 3% DSS (Fig. 3a). In accord with pH changes, the contents of lactic acid in the lesions increased dramatically post the establishment of colitis and its levels were constantly much higher in 6% DSS-treated mice than that in 3% DSS-treated littermates at the indicated timepoints (Fig. 3b). Interestingly, the levels appeared to reach the peak on day 5 in 6% DSS-instilled mice instead of 3% DSS-instilled littermates (Fig. 3b). These data indicate that the diseased colonic tissues of acute intestinal inflammation are acidic and that lower pH and more lactate accumulation was seen in the lesions of mice subjected to 6% DSS.

To further determine the acidic microenvironment of acute colitis, we detected the kinetics of SLC26A3 expression in the lesions. SLC26A3 is a transmembrane glycoprotein to transports Cl⁻ ions across the cell membrane in exchange for HCO₃⁻ as the major mechanism for regulating tissue pH^19–21. The expression of SLC26A3 decreased significantly on day 7 compared with that on day 3 in 3% DSS-instilled mice. Furthermore, its expression in 6% DSS-instilled mice was much lower than 3% DSS-instilled littermates (Fig. 3c). Intriguingly, the levels of SLC26A3 rebounded on day 7 post 6% DSS instillation (Fig. 3c), indicating a compensative response to environmental acidosis.

Next, mice were injected intraperitoneally with Cy5.5-labeled pHLIP to detect its accumulation in the inflamed colons post DSS instillation. The fluorescence was not visible in the colons of naïve mice (Fig. 3d). In contrast, pHLIP accumulated apparently in the lesions of 3% or 6% DSS-instilled mice. Furthermore, the intensity of fluorescence was significantly higher in mice drinking 6% DSS than those drinking 3% DSS (Fig. 3d). Taken together, these data indicate acidic microenvironment of acute colitis.

Pd-l1-phlip Treatment Alleviates Colitis In Mice Drinking 6% Dss

Mice drinking 3% and 6% DSS were treated with PD-L1-pHLIP at the dose of 5mg/kg, which was intraperitoneally administrated every other day. PD-L1-pHLIP treatment had no effects on weight loss and mortality in 3% DSS-instilled mice (Fig. 4a, 4c). For 6% DSS-instilled mice, weight loss in mice treated with PD-L1-pHLIP was comparable to those treated with PD-L1-Fc or PBS (Fig. 4b). However, PD-L1-pHLIP administration significantly prolonged the survival time comparable to those treated with PBS (Fig. 4d). In agreement with this, the acute colitis-triggered curtailment of colons was not obvious in PD-L1-pHLIP-treated mice compared with PD-L1-Fc or PBS-treated counterparts (Fig. 4e). Histological examination also showed that PD-L1-pHLIP treatment attenuated intestinal damage and reduced immune cell infiltration (Fig. 4f).

Targeting Dss-inflamed Colons By Fluorescent Pd-l1-phlip

We speculate that the efficacy of PD-L1-pHLIP is attributed to its targeting the lesions in response to colitis-triggered acidosis in the niche. To this end, Cy5.5-labeled PD-L1-pHLIP were injected on day 5 post the initiation of DSS instillation and fluorescent signals were detected 24 h later. The inflamed colons of 6% DSS-instilled mice were targeted well by fluorescent PD-L1-pHLIP, while there was no fluorescent signals in no-inflamed organs (i.e. kidney, lung, spleen and heart) (Fig. 5a). Intriguingly, the fluorescence was not elevated in the colons of mice drinking 3% DSS compared with naïve littermates (Fig. 5a). Significant retention of PD-L1-pHLIP in the colons of 6% DSS-instilled mice instead of 3% DSS-instilled littermates accounts for the efficacy of PD-L1-pHLIP on 6% DSS-induced colitis but not on 3% DSS-induced colitis.

PD-L1-pHLIP is well-known to work by pHLIP-mediated insertion across cellular membrane to anchor PD-L1 on the surface in response to low pH. Therefore, we determine which types of stromal cells or infiltrating immune cells were targeted by PD-L1-pHLIP. Immunofluorescence analysis of DSS-insulted colons showed that the fluorescence of PD-L1-pHLIP distributed among CK19⁺ epithelium, CD206⁺ macrophages, Ly6G⁺ granulocytes, CD11c⁺ dendritic cells and CD45⁺ leukocytes (Fig. 5b). These results thus indicate that several cell types in the niche of colitis is responsible for targeting by PD-L1-pHLIP to initiate negative immune-regulation via crosslinking PD-L1 to PD-1.

Pd-l1-phlip Treatment Reduces The Expression Of Proinflammatory Cytokines In The Lesions

The pathogenesis of colitis has been characterized by massive infiltration of immune cells and release of proinflammatory cytokines/factors in the colonic environment ^22,23. We found that, on day 6 post initiation of DSS instillation, the levels of several proinflammatory cytokines (i.e. TNF-α, IFN-γ, IL-1β, IL-17A, IL-22) decreased significantly in the colons of PD-L1-pHLIP-treated mice compared with PD-L1-Fc or PBS-treated littermates (Fig. 6a). Whereas, there was no reduction in the release of these cytokines on day 3 following PD-L1-pHLIP treatment, except for IL-1β (Fig. 6a). Notably, PD-L1-pHLIP treatment had no impact on IL-6 production (Fig. 6a).

To determine whether the efficacy of PD-L1-pHLIP was dependent on PD-L1/PD-1 signals, PD-L1-pHLIP-treated mice were injected with neutralizing anti-PD-1 antibody. Blocking PD-1 signals abrogated the therapeutic effects of PD-L1-pHLIP, as shown by curtailment of colon length in PD-1 mAb-treated mice compared with isotype-treated counterparts (Fig. 6b). Moreover, PD-1 blockade reversed reduction in colonic expression of TNF-α in PD-L1-pHLIP-treated mice (Fig. 6c). Of note, IFN-γ and IL-17A levels were not altered following PD-1 antibody administration (Fig. 6c). Considering that macrophages are predominant sources for TNF-α, which is a central mediator during intestinal inflammation²⁴, we sought to determine the effects of PD-L1-pHLIP on LPS-triggered TNF-α release in macrophages. LPS had potency to induce PD-1 expression on the surface of macrophages in a dose and time-dependent manner (Fig. 6d). Importantly, plate-coated PD-L1-pHLIP actively suppressed TNF-α production by macrophages upon exposure to LPS (Fig. 6e), which was impaired by addition of PD-1 antibody (Fig. 6f). These data indicate that PD-L1-pHLIP has capacity to inhibit TNF-α release in PD-1⁺ macrophages by delivering inhibitory signals via PD-L1/PD-1 crosslinking.

Our previous work has demonstrated that PD-L1 ectodomain tethering pHLIP potently dampened immune response to TCR stimulation under acidic conditions in vitro ¹⁰. In this study, we validated its therapeutic effects in vivo using a model of acute intestinal inflammation. PD-L1-pHLIP treatment significantly prolonged survival time of mice subjected to high-dose DSS and attenuated colonic damage by suppressing the release of proinflammatory cytokines. DSS-induced severe colitis renders lactate accumulation and acidosis in the niche, which drives presentation of PD-L1-pHLIP on the surface of epithelium and stromal cells to deliver negative signals via ligation to PD-1 and/or other unrecognized receptors (Fig. 7). Given that the soluble form of PD-L1-pHLIP has minimal effects on T cell effector function under physiological conditions, Acid-driven membrane insertion of PD-L1-pHLIP confer local immune suppression in the tissue under inflammatory attack, providing an advantage over systemically acting immune modulators.

Mechanistic investigations revealed that the efficacy of PD-L1-pHLIP was dependent on PD-L1-PD-1 engagement. Interestingly, while PD-L1-pHLIP treatment downregulated the expression of TNF-α, IFN-γ and IL-17A, PD-1 blockade just unleashed TNF-α release but not affected IFN-γ and IL-17A production. TNF-α is predominantly produced by macrophages infiltrating into the lesions and the cellular sources of IFN-γ and IL-17A are innate lymphoid cells during intestinal inflammation²⁵. In agreement with a previous study²⁶, PD-1 expression increased dramatically in macrophages upon exposure to LPS. PD-1 neutralization by mAbs abrogates the inhibition of PD-L1-pHLIP on LPS-induced TNF-α release, which suggests that PD-L1-pHLIP treatment modulates the inflammatory responses of macrophages to bacteria translocation and thereby attenuates colitis. Although PD-1 has been reported to be expressed in activated T, B and macrophages²⁷, there is no evidence that innate lymphoid cells express PD-1 to date. Notably, recent studies showed that an unrecognized receptor for PD-L1 was present and PD-L1 inhibited TNF-α production from CD11c⁺CD11b⁺ inflamed cells residing in lamina propria in a PD-1-independent fashion²⁸. It, hence, may be plausible that PD-L1-pHLIP inhibits IFN-γ and IL-17A production by innate lymphoid cells via crosslinking to an unidentified receptor, which needs further investigation in the future.

Unlike relatively constant acidity in tumor microenvironment, the acidity in the niche of inflammation is more fluctuating^19,29. We showed that local acidity in DSS-induced colitis was associated with the degree of severity and stages of inflammation, which may render inability of insertion or exit of PD-L1-pHLIP from cellular membrane when local pH is relatively high. Considering that pH₅₀ value of pHLIP utilized in this study is about 6.1, PD-L1-pHLIP may cannot be decorated on the surface of membrane at the late stage of colitis, which limits its therapeutic potentials. One approach is to utilize pHLIP variant with high pH₅₀ to conjugate PD-L1 ectodomain, for example Asp14Gla/Asp25Aad double variant (its pH₅₀ is 6.79)³⁰. This construct has been prepared and displays superior immuno-suppressive function in vitro (data not shown).

It is worthy to note that PD-L1-pHLIP had capacity to span across the membrane of several types of immune cells under acidic conditions, except for T cells. The underlying reasons are elusive. It has been proposed that the interaction between pHLIP and the membrane lipid bilayer can be affected by membrane composition^31,32, asymmetry^33–35, and the local ionic environment³⁶, using model systems of liposomes. Characterization of physical-chemical features of the membrane of T lymphocytes will thus be warranted to elucidate unresponsiveness of this population to acid-driven insertion of pHLIP. In conclusions, our work has demonstrated that PD-L1-pHLIP has potentials to be a novel cargo to treat autoimmunity and inflammatory diseases. Given that PD-L1/PD-1 signals are very crucial for brake of T cell over-activation, PD-L1-pHLIP may be more efficacious in treating T cell-trigged inflammatory diseases, such as rheumatoid arthritis and type 1 diabetes ^4,37,38. Its efficacy in murine models of these diseases is being evaluated.

Acknowledgement

This work is supported by the National Natural Science Foundation of China (81672803, 81871252).

Author Contributions

H.Z., Y.Z., H.W., J.F. and Y.S. performed the experiments and prepared the manuscript; M.Z. and X.L. were involved in optimization of the experimental protocols; H.X., C.Q., J.W., L.L., J.F. and Y.Z. provided methodological support; Y.W., Y.S and G.C. conceived and guided the study.

Conflict of interests

The authors declare no conflicts of interest.

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Acid-driven immune suppression by pHLIP-fused PD-L1 under inflammatory conditions

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Abstract

Figures

Introduction

Materials And Methods

Mouse strains, colitis model and PD-L1-pHLIP treatment

Cell And Organ Culture Preparation

Cfse Lymphocyte Proliferation Assay

Brdu Cell Proliferation Assay

Cytokine Detection By Elisa

Organ Imaging

Flow Cytometry

Measurement Of Lactic Acid Production And Ph

Histological Analysis, Immunohistochemistry And Immunofluorescence

Statistical analysis

Results

PD-L1-pHLIP inhibit T effector function in pH6.1 rather than pH6.8 buffer

Pd-l1-phlip Displays On The Surface Of Several Types Of Immune Cells Under Acidic Conditions

Low Ph And Lactate Accumulation In The Lesions Of Dss-induced Colitis

Pd-l1-phlip Treatment Alleviates Colitis In Mice Drinking 6% Dss

Targeting Dss-inflamed Colons By Fluorescent Pd-l1-phlip

Pd-l1-phlip Treatment Reduces The Expression Of Proinflammatory Cytokines In The Lesions

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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