DC-HIL/glycoprotein Nmb promotes growth of melanoma in mice by inhibiting the activation of tumor-reactive T cells - PubMed (original) (raw)

DC-HIL/glycoprotein Nmb promotes growth of melanoma in mice by inhibiting the activation of tumor-reactive T cells

Mizuki Tomihari et al. Cancer Res. 2010.

Abstract

DC-HIL/glycoprotein nmb (Gpnmb) expressed on antigen-presenting cells attenuates T-cell activation by binding to syndecan-4 (SD-4) on activated T cells. Because DC-HIL/Gpnmb is expressed abundantly by mouse and human melanoma lines, we posited that melanoma-associated DC-HIL/Gpnmb exerts similar inhibitory function on melanoma-reactive T cells. We generated small interfering RNA-transfected B16F10 melanoma cells to completely knock down DC-HIL/Gpnmb expression, with no alteration in cell morphology, melanin synthesis, or MHC class I expression. This knockdown had no effect on B16F10 proliferation in vitro or entry into the cell cycle following growth stimulation, but it markedly reduced the growth of these cells in vivo following their s.c. injection into syngeneic immunocompetent (but not immunodeficient) mice. This reduction in tumor growth was due most likely to an augmented capacity of DC-HIL-knocked down B16F10 cells (compared with controls) to activate melanoma-reactive T cells as documented in vitro and in mice. Whereas DC-HIL knockdown had no effect on susceptibility of melanoma to killing by cytotoxic T cells, blocking SD-4 function enhanced the reactivity of CD8(+) T cells to melanoma-associated antigens on parental B16F10 cells. Using an assay examining the spread to the lung following i.v. injection, DC-HIL-knocked down cells produced lung foci at similar numbers compared with that produced by control cells, but the size of the former foci was significantly smaller than the latter. We conclude that DC-HIL/Gpnmb confers upon melanoma the ability to downregulate the activation of melanoma-reactive T cells, thereby allowing melanoma to evade immunologic recognition and destruction. As such, the DC-HIL/SD-4 pathway is a potentially useful target for antimelanoma immunotherapy.

(c)2010 AACR.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1

Figure 1. Expression of DC-HIL by B16F10 melanoma and characterization of DC-HIL-knocked-down cells

Expression of DC-HIL and other co-inhibitory ligands: (A) Total RNA was isolated from B16F10 melanoma cells untreated or treated with IFN-γ (1,000 U/ml) plus PMA (10 ng/ml) for 16 h or from BM-DC and examined by RT-PCR for mRNA expression of different co-inhibitory ligands and β-actin. PCR without cDNA (No cDNA) served as negative control. Characterization of DC-HIL-knocked-down B16F10 cells: B16F10 cells transfected with control lentivector (C-B16) or DC-HIL-targeted shRNA-lentivector (Kd-B16) were assessed for protein expression (B), in vitro proliferation (C), and subjected to cell cycle analysis (D). B, Whole cell extracts prepared from B16F10 transfectants were assayed by Western blotting for protein expression of DC-HIL or β-actin using UTX-103 anti-DC-HIL or anti-β-actin Ab. C, Synchronized B16F10 transfectants (1 × 104 cells/well) were allowed to grow in vitro, and proliferation measured by MTT assay (mean ± sd, n=3). D, After starving in serum-free media, B16F10 transfectants were stimulated for growth by FCS and labeled with BrdU, and then stained with APC-anti-BrdU Ab and 7-AAD (total DNA content), followed by flow cytometry. Data are shown as dot plots of BrdU vs. 7-AAD. All data are representative of at least 2 independent experiments.

Figure 2

Figure 2. Knockdown of DC-HIL expression reduces growth of B16F10 melanoma in syngeneic wild-type (but not nu/nu) mice

Control (C-B16) or DC-HIL-knockdown B16F10 cells (Kd-B16) were inoculated s.c. into the right flank of C57BL/6 mice (12 mice/group, A and B) or nu/nu mice (5 mice/group, C), and tumor volume measured daily. Survival rate of C57BL/6 mice injected with B16F10 transfectants is shown (B). The two transfectants were also mixed (each 2 × 105 cells) and examined for tumor growth in C57BL/6 mice (n=10) (D1) and survival rate (D2). Statistical analysis (Student’s t test) of these two groups on days 13–21 indicates _p_=2 × 10−8 (A). ** (_p_= 1.5 × 10−8) and *** (_p_= 0.001) on day 24 document statistical significance of the mixed cells’ effect compared to C-B16 alone and Kd-B16 alone, respectively. Second experiment showed similar results.

Figure 3

Figure 3. Blockade of DC-HIL/SD-4 pathway leads to enhanced T cell immunogenicity

A, OVA-specific CD8+ T cells were stimulated by coculturing with OVA-pulsed/MMC-treated B16F10 transfectants and T cell activation assessed by production of IL-2 and IFN-γ. Statistical significance is denoted by * _p_=0.05 and ** _p_=0.01, as compared with production by T cells treated with C-B16 cells. B, Anti-OVA CTL were generated by immunization and subsequent in vitro stimulation and examined by flowcytometry for surface expression of SD-4 and CD69 (activation marker). C, OVA-specific CD8+ T cells were stimulated by OVA-pulsed/MMC-treated B16F10 melanoma in the absence (None) or presence of control IgG or anti-SD-4 mAb at different doses. T cell activation was assessed by IL-2 production. D, Anti-OVA CTL were allowed to kill target cells at varying E:T ratios. Target cells include OVA-pulsed Kd-B16 (• with solid lines), OVA-pulsed C-B16 (♠ with solid lines), untreated Kd-B16 (• with dashed lines), and untreated C-B16 cells (♠ with dashed lines). Statistical significance; *, _p_=0.20; **_p=_0.18, and *** _p_=0.58. Cytotoxicity is expressed as % of lysis. All data are representative of 3 separate experiments.

Figure 4

Figure 4. DC-HIL knockdown enhances capacity to stimulate anti-TAA T cells

A, Anti-hgp100/TRP-2 CTL were stimulated by C-B16 or Kd-B16 melanoma cells and IFN-γ production measured. B, These CTL were cocultured with 51Cr-labeled B16F10 transfectants or EL-4 as H-2-mismatached control, at varying E:T ratios. Statistical significance scores are denoted by * (_p_=0.59), ** (_p_=0.69), and *** (_p_=0.77). C, Two weeks after C57BL/6 mice (n=7) were tumor challenged with B16F10 transfectants, draining LN cells were prepared from treated mice and restimulated in vitro with γ-irradiated syngeneic spleen cells pulsed with hgp100 and TRP-2 peptides. Finally, IFN-γ-producing cells were counted by ELISPOT per 2 × 106 LN cells.

Figure 5

Figure 5. DC-HIL+ exsosomes from B16F10 cells inhibit T cell activation triggered by TAA on melanoma

A, Exosomes prepared from C-B16 or Kd-B16 cells were examined by immunoblotting for expression of DC-HIL and gp100 (as a melanosomal marker). B, These exosomes were added to the coculture of activated CD8+ T cells (from pmel-1 TCR-transgenic mice) and MMC-treated B16F10 cells. T cell activation was measured by production of IL-2 (left) or IFN-γ (right panel) (mean ±sd, n=3. * _p_>0.001).

Figure 6

Figure 6. Capacity of B16F10 transfectants to spread to lung and grow in the lung foci

C57BL/6 mice (n=6 for Exp 1 and n=5 for Exp 2) were given B16F10 transfectants by i.v. injection. 14 (Exp 1) or 11 (Exp 2) days post-injection, lungs were procured; weight, number of metastatic foci, total melanin content, and melanin content per focus were measured. Statistical significance (p value) is shown. Photographs of lungs from mice injected with B16F10 transfectants are shown on the right, with a scale bar of 50 mm. Images of some foci are enlarged.

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