Immune checkpoint activity regulates polycystic kidney disease progression

Innate and adaptive immune cells modulate the severity of autosomal dominant polycystic kidney disease (ADPKD), a common kidney disease with inadequate treatment options. ADPKD has parallels with cancer, in which immune checkpoint inhibitors have been shown to reactivate CD8+ T cells and slow tumor growth. We have previously shown that in PKD, CD8+ T cell loss worsens disease. This study used orthologous early-onset and adult-onset ADPKD models (Pkd1 p.R3277C) to evaluate the role of immune checkpoints in PKD. Flow cytometry of kidney cells showed increased levels of programmed cell death protein 1 (PD-1)/cytotoxic T lymphocyte associated protein 4 (CTLA-4) on T cells and programmed cell death ligand 1 (PD-L1)/CD80 on macrophages and epithelial cells in Pkd1RC/RC mice versus WT, paralleling disease severity. PD-L1/CD80 was also upregulated in ADPKD human cells and patient kidney tissue versus controls. Genetic PD-L1 loss or treatment with an anti–PD-1 antibody did not impact PKD severity in early-onset or adult-onset ADPKD models. However, treatment with anti–PD-1 plus anti–CTLA-4, blocking 2 immune checkpoints, improved PKD outcomes in adult-onset ADPKD mice; neither monotherapy altered PKD severity. Combination therapy resulted in increased kidney CD8+ T cell numbers/activation and decreased kidney regulatory T cell numbers correlative with PKD severity. Together, our data suggest that immune checkpoint activation is an important feature of and potential novel therapeutic target in ADPKD.

For all studies, both sexes, males and females, were utilized. Statistical analyses revealed no difference between males and females regarding evaluated PKD phenotypes; hence, both sexes were combined for all analyses. All animals were aged as outlined in the results sections prior to euthanasia and PKD parameter analyses were performed as outlined below. The number of animals per study varied and is indicated in the respective results sections and figure legends.

Cell Culture
All cell lines have been previously described and were originally purchased from ATCC, CRL-2833 (7).

Mouse Tissue Harvest
For all animal experiments, mice were euthanized by isoflurane exposure and cervical dislocation, and the body weight of each animal was recorded. Following, terminal heparin blood was collected via cardiac puncture. For mice analyzed by flow cytometry, mice were perfused with 20mL ice cold PBS/heparin (80units/mL) via pressure perfusion. After perfusion or directly after blood collection, the kidneys and spleen were harvested and weighed. For animals analyzed by flow cytometry, the left kidney and half the spleen were used for the single cell suspension/flow cytometry and half of the right kidney plus the other half of the spleen were fixed in 4% paraformaldehyde for histological analyses. The other half of the right kidney was flash frozen. For all other animals, the right kidney and half of the spleen were fixed in 4% paraformaldehyde for histological analyses and the left kidney plus the remainder of the spleen were flash frozen. The age of mice at euthanasia/tissue harvest varied depending on experiment (refer to results sections).

Histomorphometric and Kidney Function Analyses
Cystic index, cyst size, and cyst number were analyzed as previously published (4). In short, three kidney cross sections per animal (horizontal plane at pelvis, mid-superior and mid-inferior pole) were analyzed using a S6 custom-built NIS-Elements AR v4.6 macro (Nikon) -a cyst was defined as having a minimum feret diameter of 50µm.
Fibrotic area was analyzed from picrosirius red stained kidney sections visualized using an Olympus BX41 microscope with a linear polarizer as previously published(4). Ten random cortical 10x images were analyzed per animal.
Blood urea nitrogen (BUN) levels were analyzed using the terminal blood collection and following the manufacturer's protocol (QuantiChrom Urea Assay Kit, #501079333, BioAssay Systems). Samples were analyzed in duplicates.
C57Bl/6J Pkd1 RC/mice were treated every other day by IP injection with 10mg/kg anti-PD-1 blocking antibody (clone RMP1-14; Bio X Cell, #BP0146) or 10 mg/kg IgG2a control (clone 2A3; Bio X Cell, #BP-0089) starting at postnatal day (P) 8 until P20, at which point the animals were euthanized and evaluated as described above. For both studies, the depletion antibody and the control antibody were diluted in PBS.
Anti-PD-1/Anti-CTLA-4 study: One-month-old BALB/cJ Pkd1 RC/RC mice were treated twice a week for eight weeks by IP injection with Antibodies were diluted in InVivoPure pH 7.0 Dilution Buffer (Bio X Cell, #IP0070). After eight weeks of treatment, the animals were euthanized and evaluated as described above. Supplemental Table 4 shows general S7 phenotyping characteristics of one-month-old BALB/cJ Pkd1 RC/RC mice, providing a baseline of ADPKD severity at time of treatment start. These parameters were obtained from separate animals not included in the anti-PD-1/anti-CTLA-4 study.
The chosen dose of anti-PD-1 and anti-CTLA-4 is within the range that is clinically given, and the doses given as well as the frequency has been critically tested in multiple murine models(9-15).

Single Cell Suspension & Flow Cytometry
Single cell suspensions of the dissected kidneys were prepared as previously described (4,8). In short, Gating for PD-L1 positive epithelial cells and macrophages was done as follows and as outlined in (8): Live singlet cells were gated for CD45 positive or CD45 negative. CD45 negative cells were gated for being either APN + or EpCAM + using a Boolean "OR" gate (epithelial cells). Epithelial cells were then analyzed to be either positive or negative for PD-L1 staining. A FMO sample was used to set the negative gate ( Figure 1E).
Macrophages were defined by analyzing CD45 + cells that were CD19and Ly6G -, to exclude B cells and neutrophils. Resulting cells were then gated for CD64 + events, followed by analysis of PD-L1 positive expression using the same gate used for epithelial cells (Figure 1C).

Study Approval
All experimental procedures were performed in an AAALAC-accredited facility in accordance with the

Guide for the Care and Use of Laboratory Animals(16) and approved by the University of Colorado Anschutz
Medical Campus Institutional Animal Care and Use Committee (protocols #33, 301, 685).

Data Availability
All underlying data generated as part of this manuscript are available from the corresponding author upon request and an MTA in place. S11

SUPPLEMENTAL TABLES
Supplemental