Sources of antibodies

The following antibodies were purchased from BD Biosciences (Waltham, Massachusetts, USA): anti-CD45 (AF700, clone: 30-F11), anti-CD3 (BV421, clone: 145-2 C11), anti-CD4 (BV510, clone: RM4-5), anti-CD8 (PE, clone: 53-6.7), anti-NK1.1 (PE-CY7, clone: PK136), anti-CD11b (BV605, clone: M1/70), anti-CD11c (APC, clone: HL3), anti-PD-1 (BV650, clone: J43), anti-PD-L1 (BV650, clone: MIH5), anti-FoxP3 (AF647, clone: MF23), anti-IFN-γ (AF647, clone: XMG1.2), anti-Ki-67 (AF488, clone: B56), anti-granzyme B (eFluor 660, clone: NGZB), anti-CD80 (BV421, clone: 16-10 A1), anti-CD86 (BV450, GL1), and anti-TNF-α (AF488, clone: MP6-XT22). Additionally, anti-PD-1 (clone: RMP1-14), anti-PD-L1 (clone: 10F.9G2), anti-CD4 (clone: GK1.5), anti-CD8 (clone:2.43), and anti-NK1.1 (clone: PK136) were purchased from BioXcell (Lebanon, USA).

Cell lines and culture conditions

The cell lines were purchased from American Type Culture Collection (ATCC, Manassas, Virginia, USA), including murine B16-F10 melanoma cells (CRL-6475), murine Lewis lung carcinoma LLC (CRL-1642), murine colon adenocarcinoma cells MC38 (Kerafast ENH204-FP), and human foreskin fibroblasts (HFFs) (SCRC-1041). Murine melanoma B16-F10 labeled with luciferase (B16-F10-Luc) was generated as described previously.42 These cell lines were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS; Gibco, New York, USA) and 1% penicillin-streptomycin-glutamine 100x (Thermo, Waltham, Massachusetts, USA). HFFs were used to culture T. gondii ΔGRA17 tachyzoites. Lymphocytes were grown in RPMI 1640 medium containing 10% of HI-FBS, 10 mM HEPES, 1 mM sodium pyruvate, 0.05 mM β-mercaptoethanol, 1% penicillin–streptomycin–glutamine, and 1× minimal essential medium non-essential amino acids. Lymphocytes were used for flow cytometry analysis of tumor-infiltrating T cells and the immune status of the injected and uninjected tumors. Cultures of all above-mentioned cell lines and T. gondii were maintained in a humidified atmosphere at 37°C in 5% CO₂.

Mice

Six to 8 week-old, female C57BL/6 mice and NOD/SCID/IL-2R g-chain knockout mice (NSG) were purchased from Zhejiang Laboratory Animal Center, Hangzhou, China. All mice were maintained on a standard commercial diet and sterile water ad libitum throughout the experiments. Mice were housed under specific-pathogen-free conditions and a 12 hour/12 hour light–dark cycle. Mice were randomly assigned to experimental groups. The study protocols were reviewed and approved by the Research Ethics Committee of Ningbo University (Permit No. SYXK (ZHE) 2019-0005). Mice were observed two times per day and were euthanized once they have reached their humane endpoint, including 20% loss of body weight from tumor induction, body condition score ≤2, or when the tumor size reaches ~1.5 cm.

Construction of T. gondii RH ΔGRA17 mutant strain

Type I T. gondii RH ΔGRA17 strain was constructed using CRISPR-Cas9 system as previously described.41 Deletion of GRA17 gene in the virulent RH strain diminishes the parasite’s ability to proliferate in vitro and partly attenuates its virulence in mice.41 Tachyzoites of ΔGRA17 strain were stored frozen at −80°C until analysis. Prior to administration of ΔGRA17, tachyzoites were purified by filtration using a 3.0 µm filter (Nuclepore; Sterlitech, Kent, Washington, USA) and washed with DMEM culture medium. The viability and number of ΔGRA17 tachyzoites were determined using trypan blue exclusion assay and cell counting with a hemocytometer, respectively.

Optimal dosing of T. gondii ΔGRA17

We performed a titration experiment to determine the optimal dose of tachyzoites for examining the therapeutic efficacy of intratumoral administration of ΔGRA17 strain. Briefly, mice bearing B16-F10 tumors were intratumorally injected with phosphate buffered saline (PBS) or serial doses of ΔGRA17 tachyzoites (5×10³, 1×10⁴, 5×10⁴, or 1×10⁵) at days 9, 11, and 13 after subcutaneous implantation of B16-F10 cells in the right flank of mice. A digital caliper was used to determine the volume of the subcutaneous tumors. The tumor volumes were calculated using the formula (width² ×length)/2.

Efficacy of T. gondii ΔGRA17 against B16-F10 melanoma

We established a unilateral B16-F10 melanoma mouse model. Briefly, 1×10⁶ to 3×10⁶ B16-F10 cells were subcutaneously inoculated into the right flank of mice. When the tumor’s size has reached ~0.5 cm (at ~9 days), 100 µL of PBS or 5×10⁴ ΔGRA17 tachyzoites were intratumorally injected on days 9, 11, and 13. We then determined the antitumor activity of ΔGRA17 treatment on the established unilateral B16-F10 melanoma by analyzing the tumor volume using a digital caliper, starting from day 9 and every other day until the end of the experiment (ie, 19 days after B16-F10 cell implantation). The tumors were dissected out after euthanasia of the mice and the tumor’s size was compared between ΔGRA17-treated and PBS-treated (control) mice. The body weight of ΔGRA17-treated and control mice was monitored two times per day up to 30 days.

The expression of mouse melanoma markers, CD63, S100β, and Ki67,43 was studied using immunohistochemistry analysis. Briefly, following deparaffinization and rehydration of the paraffin-embedded tumor tissue sections, the antigen was retrieved with high temperature in sodium citrate solution 0.01 M, pH 6.0. Then, histological sections were incubated with diaminobenzidine and counterstained with hematoxylin (Solarbio, Beijing, China), followed by incubation with anti-CD63, anti-S100β, and anti-Ki67 and antibodies (Servicebio, Wuhan, China), and secondary antibodies (Servicebio, Wuhan, China). Images were acquired using a microscope with ×200 magnification (Olympus, Tokyo, Japan).

Immunohistochemical (IHC) staining of treated and untreated tumor tissue was scored by two independent pathologists blinded to the mouse data according to the semiquantitative Immunoreactivity Score (IRS). Category A documented the percentage of stained area with immunoreactive cells as 0 (0%–5%), 1 (6%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (76%–100%). Category B documented the intensity of immunostaining as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). Multiplication of category A and B resulted in an IRS ranging from 0 to 12.

The survival of ΔGRA17-inoculated and PBS-inoculated (control) mice was monitored for up to 30 days. Mice that remained tumor-free after 30 days from B16-F10 induction were rechallenged with B16-F10 cells in the opposite (left) flank. These mice were monitored for additional 60 days (ie, a total of 90 days after tumor implantation) for the development of the reimplanted tumors.

Analysis of key parameters of the biologic feasibility of ΔGRA17

To test the therapeutic efficacy of ΔGRA17 injected into extratumoral sites, we injected mice with 100 µL of PBS or 5×10⁴ ΔGRA17 intratumorally, intraperitoneally, or intravenously. The tumor volume in mice inoculated by each of these three different inoculation routes was determined up to 19 days and mouse survival was determined up to 30 days.

To determine whether latent toxoplasmosis interferes with treatment efficacy, we tested the efficacy of ΔGRA17 against B16-F10 melanoma in mice chronically infected via intragastric administration with 10 cysts of T. gondii (Type II) PRU strain. The tumor volume was determined up to 17 days and mouse survival was determined up to 30 days.

We investigated whether live ΔGRA17 tachyzoites are necessary to achieve the required antitumor efficacy or ΔGRA17-derived constituents are sufficient to elicit an adequate immune response via recognition of the parasite’s molecular patterns. Briefly, tachyzoites obtained from viable ΔGRA17 strain or inactivated ΔGRA17 strain, prepared by heating to 60℃ for 3 min, were inoculated into B16-F10 melanoma-bearing mice as described above. The tumor volume in mice inoculated by viable ΔGRA17 tachyzoites, inactivated ΔGRA17 tachyzoites, or PBS was determined up to 19 days, and mouse survival was determined up to 30 days.

We further used immunocompromised NSG mice bearing B16-F10 tumors to determine whether an active immune response is necessary for ΔGRA17-mediated clearance of pre-existing B16-F10 tumors. The tumor volume in NSG mice inoculated by ΔGRA17 tachyzoites or PBS was determined up to 16 days, and mouse survival was determined up to 20 days.

Tumor-infiltrating T cells (TILs) after administration of ΔGRA17

The levels of expression of key proinflammatory cytokines IFN-γ (70-EK280HS), interleukin (IL)−12p40 (70-EK2183), and IL-12p70 (70-EK212HS) in the tumor tissue of ΔGRA17-inoculated mice 24 hours after the second treatment (ie, on day 12) were determined using ELISA kits as per the manufacturer’s instructions (Multisciences, Hangzhou, China). We also analyzed intratumoral immune cell infiltration by using flow cytometry. Briefly, tumors were dissected out of the mouse tissue and digested with 2% FBS, collagenase type I (Clone: SCR103, Sigma), and DNase I (Clone: H6254, Sigma) in RPMI 1640 medium for 1 hour at 37°C. The tumor tissues were homogenized and filtered through a 70 µm nylon strainer to obtain single cell suspensions. The levels of immune cells expressing CD45⁺, CD3⁺, CD4⁺, CD8⁺, NK1.1, CD11b⁺, CD11c⁺, IFN-γ, TNF-α, Ki-67, and granzyme B were analyzed using Beckman CytoFLEX S and FlowJo software (Tree Star, Ashland, Oregon, USA).

Mouse (IFN-γ enzyme-linked immune absorbent spot (ELISPOT) assay was used to monitor IFN-γ secretion by tumor-infiltrating T cells as described previously.26 Briefly, CD8⁺ T cells were isolated from spleen or draining inguinal lymph nodes and purified using anti-CD8 MACS magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD8⁺ T cells were cocultured at 10:1 ratio with cell targets (irradiated EL-4 thymoma cell) for 48 hours in coated and blocked ELISpot plates. Cell targets were previously pulsed overnight with 1 mg/mL MHC-I–restricted peptide epitopes TRP-2 (melanoma Ag), ovalbumin (OVA), or heat-killed T. gondii ΔGRA17 Ag. Control samples were pulsed with culture media. IFN-γ–expressing T cells were detected and analyzed according to the manufacturer’s protocol (BD Bioscience).

We examined whether treatment efficacy could be extended to other tumor types via intratumoral administration of ΔGRA17 in mouse MC38 colon carcinoma and LLC lung carcinoma models. Briefly, a similar treatment schedule was followed as described above, except that MC38 or LLC tumor cells, instead of B16-F10 tumor cells, were injected in the right flank of mice. MC38 and LLC tumor volume was determined. We also studied the intratumoral immune infiltrates in LLC or MC38 tumor tissues. We determined the percentages of CD45⁺ immune cells, CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophages and DCs in PBS-treated or ΔGRA17-treated LLC tumor cells and PBS-treated or ΔGRA17-treated MC38 tumor cells.

Systemic effects of ΔGRA17 on distant tumors

We investigated whether ΔGRA17 treatment has a systemic immune-enhancing effect on non-injected (distant) tumors. To establish a bilateral tumor model, 1×10⁶ to 3×10⁶ of B16-F10, LLC or MC38 cells were subcutaneously inoculated into the right flank of mice. Three days later, 5×10⁵ to 8×10⁵ cells of each cell line were subcutaneously inoculated into the left flank. Then, 5×10⁴ ΔGRA17 tachyzoites were inoculated inside the right flak tumor as described above. We determined the volume of the inoculated and non-inoculated (distant) tumors over 29–31 days and monitored the overall mouse survival in the bilateral B16-F10, LLC, or MC38 tumor models over 40–50 days.

To detect the tumorigenesis of mouse melanoma, B16-F10-Luc cells were used to establish B16-F10-Luc tumors, and the tumor size was monitored by bioluminescence imaging using PerkinElmer IVIS Lumina XRMS instrument (PerkinElmer, Waltham, USA). Immediately before the in vivo imaging, mice received intraperitoneally 200 µL of VivoGlo fluorescein (15 mg/mL) (Promega, Madison, USA). Additionally, IHC staining and IRS analysis of mouse tumor tissues were performed as described above to determine the expression of CD63, S100β, and Ki67 in melanoma tissue. Furthermore, systemic therapeutic efficacy of ΔGRA17 was determined by comparing the volume of the injected and distant tumor (ie, MC38 colon carcinoma and the LLC lung carcinoma) and by monitoring the survival of mice.

Analysis of the immune status of the injected and distant tumors

We examined whether intratumoral administration of ΔGRA17 changes the immune status of non-injected (distant) tumors. A treatment schedule was followed as described in figure 1A, and B16-F10 tumors were harvested 24 hours after the second treatment (ie, on day 12). Dissected tumors were digested for preparation of single-cell suspensions followed by antibody staining for CD45⁺, CD3⁺, CD4⁺, CD8⁺, NK1.1, CD11b⁺, CD11c⁺, IFN-γ, TNF-α, Ki67, and granzyme B. Cytometry analysis and IFN-γ ELISPOT assay were performed as described above. The numbers of infiltrating immune cells in treated tumors and distant, untreated, tumors were determined and compared.

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Figure 1

Toxoplasma gondii ΔGRA17 strain significantly reduces the growth of melanoma in mice. (A) Schematic representation of the treatment regimen and timeline for mice inoculated with B16-F10 cells in the right flank. Once the B16-F10 tumor size has reached 5 mm at ~9 day, the tumor was injected with 100 µL of phosphate buffered saline (PBS) (control) or 5×10⁴ ΔGRA17 tachyzoites and then again at days 11 and 13 after implantation of B16-F10 cells. Using a vernier caliper, the tumor size was measured, starting from day 9 and every other day until 19 days after B16-F10 cells’ implantation. When the tumor size has reached ~15 mm, mice were humanely euthanized. (B) ΔGRA17 administration significantly reduced the tumor size compared with control mice treated with PBS only. (C) The size and location of the tumors detected in mice euthanized on day 19. (D) The tumors dissected from mice shown in (C), showing a significant reduction in the tumor size of ΔGRA17-injected mice compared with control mice. (E) Immunohistochemical analysis and the corresponding Immunoreactivity Score (IRS) of mouse melanoma markers CD63, S100β, and Ki67. (F) Survival curve of B16-F10-bearing mice shows a significant improvement in the survival of ΔGRA17-inoculated mice compared with control mice. (G) Tumor-free mice were reinoculated in the left flank with 1×10⁶ to 3×10⁶ B16-F10 cells at 30 days and their survival was monitored for 60 days (ie, 90 days after tumor implantation). The survival of tumor-bearing mice was presented as Kaplan-Meier plots and tested for significance using log-rank tests. Data are mean±SD (n=6 mice/group) of three independent experiments; unpaired Student’s t-test, *p<0.05, **p<0.01, ***p<0.001, n/s, not significant, compared with the indicated control.

To determine whether the regression of distant tumors was related to the immune responses evoked by TILs or to the systemic dissemination of ΔGRA17, we analyzed T. gondii-specific B1 gene expression of ΔGRA17 tachyzoites using quantitative PCR (qPCR). Briefly, tumor cells were injected into both flanks of wild type (WT) mice and NSG mice. On day 9, the right flank was injected with 5×10⁴ ΔGRA17 tachyzoites, and 5 days later (ie, on day 14), the injected and the distal tumors were harvested from WT and NSG mice. Tumors were homogenized using the gentle MACS Dissociator (Miltenyi Biotec, Bergisch Bladbach, Germany) and genomic DNA was isolated using a DNA extraction Kit (TIANGEN, Beijing, China). The isolated DNA was used as a template to amplify T. gondii B1 gene. qPCR was performed using a SYBR Premix Ex Taq II (Takara, Tokoyo, Japan) on a Mx3005P real-time PCR System (Stratagene, Waltham, Massachusetts, USA) as described previously.44

We established a standard curve to quantify the number of ΔGRA17 tachyzoites from the Ct values obtained from qPCR analysis of tumor tissues. Briefly, DNA was isolated from a 10-fold dilution series of ΔGRA17 tachyzoites (10⁶, 10⁵, 10⁴, 10³). qPCR was carried out as described above. This experiment was repeated three times, and mean Ct values were used to generate a standard curve (R2=0.99, E=98%, y=−2.252x+31.02) by plotting the Ct values against the logarithm of the numbers of tachyzoites in each dilution. Linear regression analysis was used to fit the curve to the data. PCR amplification efficiency (E) was calculated from the slope of the standard curve using the equation E = [10^−1/slope−1].

Profiling immune-related genes in treated and distant tumors of ΔGRA17-treated mice

We further examined whether intratumoral administration of ΔGRA17 changes the immune status of the distant tumors in B16-F10-bearing mice. NanoString platform (WuXi AppTec, Shanghai, China) was used to study the gene expression of the injected tumors, distant tumors, and control mouse groups. Briefly, B16-F10 tumor-bearing mice were treated with PBS or ΔGRA17 as described above. Twenty-four hours after the second treatment (ie, on day 12), RNA was isolated from the dissected tumors and hybridized with the PanCancer Mouse Immune Profiling panel and quantified using the digital analyzer (nCounter). Data were processed and normalized using nSolver Analysis Software and the Advanced Analysis module (NanoString Technologies, Waltham, Massachusetts, USA). The expression profile of immune-related pathways was determined in the injected and distant tumors. The ggpubr R package was used to construct the volcano plots and pheatmap R package was used to perform hierarchical clustering of tissue gene expression.

The expression levels of mRNA of genes (H2-K1, CD74, H2-Eb1, H2-T23, H2-Aa, Psmb8, CCL3, IL1b, CD80, Fas, IL2RA, and JAK3) involved in antigen processing and T cell function in the injected and distant tumors were determined by quantitative reverse transcriptase PCR (qRT-PCR). Briefly, total RNA was isolated from B16-F10 tumor tissue using Trizol reagent (Invitrogen, Waltham, Massachusetts, USA). DeNovix DS-11 spectrophotometer (DeNovix, Waltham, Massachusetts, USA) was used to detect the purity and concentration of RNA. qRT-PCR analysis was performed using SYBR Premix Ex Taq II (Takara, Tokyo, Japan) with a Mx3005P real-time PCR System (Stratagene, La Jolla, California, USA). The primer sequences are provided in the online supplemental material.

Immunofluorescence analysis was also used to quantify the level of expression of PD-L1 in injected and distant tumors. Briefly, formalin-fixed, paraffin-embedded B16-F10 tumor tissue was cut into 6 µm. The sections were deparaffinized in xylene and rehydrated in a graded series of ethanol. For antigen retrieval, the sections were immersed in 10 mM sodium citrate (pH 6.0), heated in a water bath at 95°C–99°C for 20 min, and then cooled to room temperature. Then, slides containing B16-F10 tumor tissue were fixed for 10 min at 4°C in 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, USA) for 10 min at room temperature, and blocked for 1 hour in PBS with 1% horse serum (Sigma-Aldrich, Saint Louis, USA) overnight at 4°C and washed three times for 5 min with PBS. Slides were subsequently incubated with primary antibodies: anti-PD-L1 (1:100; clone: ab213524, Abcam, Cambridge, UK) and anti-Firefly-Luciferase (1:100, clone: ab21176, Abcam, Cambridge, UK). Alexa Fluor secondary antibody (1:500; ab150077, Abcam, Cambridge, UK) was added for 1 hour at room temperature in the dark. The slides were washed three times with PBS and DAPI (Sigma-Aldrich, Saint Louis, USA) was added to the first wash to stain the nuclei. Images were acquired using a Confocal Zeiss LSM880. Intensity analysis was performed using ImageJ V.1.51j8 (National Institutes of Health, USA).

To further examine how PD-L1 expression is influenced in B16-F10 cells following ΔGRA17 infection, B16-F10 cells cultured in vitro were PBS treated (control) or infected with ΔGRA17 tachyzoites and harvested after 24 hours. The cells were then blocked with a-CD16/32 Ab (clone 93, eBioscience; 1:1000) and stained with antibodies against mouse PD-L1. The cell surface expression levels of PD-L1 were shown as mean fluorescence intensity (MFI) values.

We examined whether T. gondiiΔGRA17 treatment increased expression of CD80 in the tumor microenvironment. CD45⁺CD11c⁺ cells from ΔGRA17-injected and distant B16-F10 tumor-bearing mice were analyzed for the expression CD80⁺ using flow cytometry.

Combination therapy using ΔGRA17 and PD-L1 blockade

To examine the efficacy of intratumoral therapy using ΔGRA17 and PD-L1 on bilateral (injected and distant) melanomas, B16-F10-bearing mice were treated with ΔGRA17 on one side in addition to intraperitoneal injection with or without 250 µg anti-PD-L1 antibody on days 9, 11, and 13. Control mice included age-matched B16-F10-bearing mice treated with PBS. The tumorigenesis or regression of mouse B16-F10-Luc cells was determined using bioluminescence imaging as described above. The tumor volume (up to 30 days) and survival (up to 60 days) of mice receiving monotherapy or dual therapy was determined. Body weight in each mouse group was also determined. Mice were deemed to be completely cured when no tumor was detected by palpation. Mice cured of B16-F10 tumors and age-matched tumor-naive (control) mice were rechallenged with the same tumors within 25 days. The previously cured mice were treated with a combination therapy (ΔGRA17 and PD-L1 blockade) in the right flank on day 60 to determine the long-term persistence of antitumor memory, while control mice received no treatment.

Role of immune cells in the efficacy of the dual ΔGRA17 plus PD-L1 blockade therapy

We determined which cellular subsets underlay the observed therapeutic effect of the combined therapy and investigated the role of cellular subsets during early and late stage of dual therapy. Briefly, mice were injected intraperitoneally with 500 µg monoclonal antibodies (Ab) to CD8⁺, CD4⁺, or NK1.1, either the day before tumor implantation (day –1) or on the first day of treatment (day 0), followed by injections of 250 µg every 5 days until mice have reached their humane endpoint. Animals that did not receive any depleting Ab for CD4⁺, CD8⁺, and NK cells were used as controls. The survival of mice was monitored two times per day.

Statistical analysis

Statistical analysis was performed using GraphPad Prism V.8 (GraphPad Software, California, USA). Two-tailed Student’s t-test was used to derive the significance of the differences between the two groups. Differences were considered significant when p<0.05. We used log-rank tests for the Kaplan-Meier survival analysis. The numbers of mice used in each experiment are provided in the figure legends. Data are presented as mean±SD. All experiments were performed in triplicate.