CD1d-expressing breast cancer cells modulate NKT cell-mediated antitumor immunity in a murine model of breast cancer metastasis

Abstract

Background: Tumor tolerance and immune suppression remain formidable obstacles to the efficacy of immunotherapies that harness the immune system to eradicate breast cancer. A novel syngeneic mouse model of breast cancer metastasis was developed in our lab to investigate mechanisms of immune regulation of breast cancer. Comparative analysis of low-metastatic vs. highly metastatic tumor cells isolated from these mice revealed several important genetic alterations related to immune control of cancer, including a significant downregulation of cd1d1 in the highly metastatic tumor cells. The cd1d1 gene in mice encodes the MHC class I-like molecule CD1d, which presents glycolipid antigens to a specialized subset of T cells known as natural killer T (NKT) cells. We hypothesize that breast cancer cells, through downregulation of CD1d and subsequent evasion of NKT-mediated antitumor immunity, gain increased potential for metastatic tumor progression.

Methodology/principal findings: In this study, we demonstrate in a mouse model of breast cancer metastasis that tumor downregulation of CD1d inhibits iNKT-mediated antitumor immunity and promotes metastatic breast cancer progression in a CD1d-dependent manner in vitro and in vivo. Using NKT-deficient transgenic mouse models, we demonstrate important differences between type I and type II NKT cells in their ability to regulate antitumor immunity of CD1d-expressing breast tumors.

Conclusions/significance: The results of this study emphasize the importance of determining the CD1d expression status of the tumor when tailoring NKT-based immunotherapies for the prevention and treatment of metastatic breast cancer.

Figure 1. Decreased expression of CD1d in highly metastatic murine and human breast cancer cells.

(A) Real-time RT-PCR assay confirming significant downregulation of the CD1d1 gene in TM40D-MB cells, as compared to parental TM40D (low metastatic) cells. L19 serves as an internal control. Experiments were performed in triplicate, and data are represented as the mean ± SEM, * P≤0.05. (B) Flow cytometry analysis of CD1d using a PE-conjugated anti-CD1d mAb (1B1) or isotype IgG2b control. Average ± SD mean fluorescence intensity (MFI) for three independent experiments: TM40D (blue)  = 928.7±21.2, TM40D-MB (red)  = 541.0±57, ** P≤0.001. (C) Decreased expression of CD1d in human mammary epithelial cells correlates with increasing metastatic potential by RT-PCR. 71N, 81N: normal transformed human mammary epithelial cells. 21PT, ZR75: primary breast adenocarcinoma cells. MCF-7: minimally invasive adenocarcinoma cells. MDA-MB-468, MDA-MB-231: highly metastatic human breast adenocarcinoma cells, from patients of African-American (MDA-MB-468) and Caucasian (MDA-MB-231) descent. GAPDH serves as an internal housekeeping gene control.

Figure 2. Increased tumor cytolysis of CD1d-expressing cells by enriched iNKT cells.

(A) FACS analysis of enriched iNKT cells by magnetic bead sorting using PE-conjugated PBS-57-loaded CD1d tetramer and FITC-conjugated anti-TCRβ Ab. Activation assessed using PerCP-conjugated anti-CD69 Ab. Mean fluorescence intensity (MFI) for control naïve CD4⁺ T Cell = 99 (black), Unenriched iNKT = 121 (blue), Enriched iNKT = 715 (red). (B) In vitro Lactate Dehydrogenase (LDH) cytotoxicity assay. Positively-enriched iNKT effector cells (>20% iNKT+) were incubated with TM40D or TM40D-MB target cells at E:T ratios of 5∶1, 10∶1 and 25∶1, for 4 hrs at 37°C, 5% CO₂. Cell-free supernatants were assayed for LDH activity as a measure of cell lysis. (C) TM40D target cells were incubated with enriched iNKT cells at E:T ratios of 5∶1, 10∶1 and 25∶1, in the presence or absence of anti-CD1d (3C11) blocking antibody (10 µg/ml). (D) In vitro anti-CD1d antibody titration in TM40D cells. Positively-enriched iNKT effector cells (>20% iNKT⁺) were incubated with TM40D target cells at an E:T ratio of 25∶1, in the presence of anti-CD1d (3C11) blocking antibody or IgM isotype control, at the concentrations indicated. Data are presented as mean ± SD, * P<0.05. Data are representative of at least two independent experiments.

Figure 3. Decreased CD1d expression by tumor correlates with decreased iNKT-mediated antitumor immunity in vivo.

(A) Orthotopic injection into the bilateral mammary fat pads of mice with either 1×10⁶ CD1d-expressing TM40D or CD1d-deficient TM40D-MB cells, 5 mice per tumor group. Mice were monitored for tumor formation (tumor size of 0.3 cm), and mice in each tumor group were euthanized at maximum tumor size (2 cm). (B) FACS analysis comparing spleens isolated from either TM40D or TM40D-MB tumor-implanted mice. Splenocytes were isolated from mice at maximum tumor size and analyzed by FACS for live iNKT cell populations using APC-conjugated PBS-57-loaded CD1d tetramer and FITC-conjugated anti-TCRβ antibodies. Live NK populations were assessed using PE-conjugated anti-CD49b (DX5) and FITC-conjugated anti-TCRβ antibodies. Live CD4⁺ and CD8⁺ T cell populations were assessed using APC-conjugated anti-CD4 and PerCP-Cy5.5-conjugated anti-CD8α antibodies. C,(D) Histograms quantifying the total percentage of live iNKT, NK, CD4⁺ and CD8⁺ T cell of total splenocytes. N = 3 (Unchallenged). N = 5 (TM40D, TM40D-MB). Data are mean ± SD. * P≤0.05, ** P≤0.001. These results are representative of at least two independent experiments.

Figure 4. Antibody blocking of CD1d-expressing TM40D tumor cells increases spontaneous lung metastasis in vivo.

(A) Comparison of tumor growth in mice administered anti-CD1d blocking antibody or vehicle control. Wild-type BALB/c mice (5 mice per group) were implanted with TM40D tumor cells and inoculated I.P. with either with 200 µg of anti-CD1d (3C11) blocking antibody or vehicle control (TM40D-C) at days 10, 17, 24, and 31 post tumor implantation. Mice were monitored for tumor formation (tumor size of 0.3 cm), and mice were euthanized at maximum tumor size (2 cm). (B) In vivo anti-CD1d antibody blockade of CD1d-expressing TM40D tumors increases the frequency of lung tumor metastases in mice. At maximum tumor volume, lung tissues were isolated and fixed in Bouin's Fixative and scored for visible metastasis foci under dissecting light microscope. (C) Scatter plot depicting average number of tumor foci counted per lung (** P<0.005, one-way ANOVA test).

Figure 5. Gene knockdown of CD1d by shRNA in CD1d-expressing TM40D cells suppresses in vivo iNKT-mediated antitumor immunity and promotes increased spontaneous metastasis to lung.

(A) TM40D cells were transduced by a pLKO.1 lentivirus expressing shRNA against murine cd1d1. Real-time RT-PCR assay confirming downregulation of the cd1d gene in the TM40D-shCD1d cells, as compared to parental TM40D and TM40D-MB cells and scrambled shRNA control (TM40D-scr). TM40D-MB cells, as compared to parental TM40D (low metastatic) cells. GAPDH serves as an internal control. (B) FACS analysis of gene knockdown of CD1d using a PE-conjugated anti-CD1d mAb (1B1) or isotype IgG2b control. Mean fluorescence intensity (MFI) for IgG2b Isotype Control (black)  = 127, TM40D-shCD1d (green)  = 389, TM40D-MB (red)  = 599, TM40D (blue)  = 932. (C) Suppression of iNKT-regulated lymphocytes in vivo of TM40D-shCD1d tumor-implanted mice, as compared to parental TM40D control. Histograms quantifying the total percentage of live iNKT, and CD4⁺ and CD8⁺ T cell of total splenocytes. N = 5 (TM40D, TM40D-shCD1d). Data are mean ± SD. * P≤0.05. (D) Scatter plot depicting increased number of tumor foci counted per lung in TM40D-shCD1d tumor-implanted mice as compared to TM40D parental control. (* P≤0.05, one-way ANOVA test).

Figure 6. Differences in tumor growth and metastasis between TM40D and TM40D-MB tumors in normal and immune deficient mice.

Orthotopic injection into the bilateral mammary fat pads of CD1d KO, Jα18 KO, RAG2 KO, or wildtype BALB/c mice, with either 1×10⁶ TM40D (CD1d-hi) or TM40D-MB (CD1d-lo) cells, 5 mice per tumor group. Mice were monitored for tumor formation (tumor size of 0.3 cm), and mice in each tumor group were euthanized at maximum allowable size (2 cm). (A) Comparison of TM40D (CD1d-hi) tumor growth in CD1d KO, Jα18 KO, RAG2 KO, or wildtype BALB/c mice. (B) Differences in rates of tumor growth and metastasis between TM40D and TM40D-MB tumors in normal and immune deficient mice. (C) Comparison of TM40D-MB (CD1d-lo) tumor growth in CD1d KO, Jα18 KO, RAG2 KO, or wildtype BALB/c mice. Data are mean ± SD. (D) Scatter plot depicting increased number of tumor foci counted per lung in TM40D or TM40D-MB tumor-implanted mice in immune-deficient CD1d KO, Jα18 KO, RAG2 KO, or wildtype BALB/c mice. (* P≤0.05, ** P<0.005, *** P≤0.001, one-way ANOVA test). Data are representative of two independent experiments.