Disrupted lymph node and splenic stroma in mice with induced inflammatory melanomas is associated with impaired recruitment of T and dendritic cells

International audienceMigration of dendritic cells (DC) from the tumor environment to the T cell cortex in tumor-draining lymph nodes (TDLN) is essential for priming naïve T lymphocytes (TL) to tumor antigen (Ag). We used a mouse model of induced melanoma in which similar oncogenic events generate two phenotypically distinct melanomas to study the influence of tumor-associated inflammation on secondary lymphoid organ (SLO) organization. One tumor promotes inflammatory cytokines, leading to mobilization of immature myeloid cells (iMC) to the tumor and SLO; the other does not. We report that inflammatory tumors induced alterations of the stromal cell network of SLO, profoundly altering the distribution of TL and the capacity of skin-derived DC and TL to migrate or home to TDLN. These defects, which did not require tumor invasion, correlated with loss of fibroblastic reticular cells in T cell zones and in impaired production of CCL21. Infiltrating iMC accumulated in the TDLN medulla and the splenic red pulp. We propose that impaired function of the stromal cell network during chronic inflammation induced by some tumors renders spleens non-receptive to TL and TDLN non-receptive to TL and migratory DC, while the entry of iMC into these perturbed SLO is enhanced. This could constitute a mechanism by which inflammatory tumors escape immune control. If our results apply to inflammatory tumors in general, the demonstration that SLO are poorly receptive to CCR7-dependent migration of skin-derived DC and naïve TL may constitute an obstacle for proposed vaccination or adoptive TL therapies of their hosts

Tumor-initiated inflammation overrides protective adaptive immunity in an induced melanoma model in mice

We studied the effect of the immune system on two differentially aggressive melanomas developing in mice on conditional deletion of the INK4A/ARF tumor suppressor gene, with concomitant expression of oncogene H-Ras(G12V) and a natural cancer-germline tumor antigen (TA). "Slow progressor" melanomas contained no activated T lymphocytes (TL). In contrast, "aggressive" melanomas were infiltrated by activated TLs lacking effector molecules and expressing high levels of PD-1, indicating an exhausted phenotype. Aggressive melanomas were also infiltrated by immature myeloid cells (IMC). Infiltration was associated with local inflammation and systemic Th2/Th17-oriented chronic inflammation that seemed to impair further activation of TLs, as tumor-specific T cells adoptively transferred into mice bearing aggressive melanomas were poorly activated and failed to infiltrate the melanoma. This immunosuppression also led to the incapacity of these mice to reject inoculated TA-positive tumors, in contrast to slow-progressing melanoma-bearing mice, which were responsive. To test the role of adaptive immunity in tumor progression, we induced melanomas in immunodeficient RagKO compound mice. These mice developed aggressive but not slow-progressing melanomas at a higher frequency and with a shorter latency than immunocompetent mice. Immunodeficient mice also developed abnormal inflammation and infiltration of IMCs in a manner similar to immunocompetent mice, indicating that this phenotype was not dependent on adaptive immunity. Therefore, tumor-intrinsic factors distinguishing the two melanoma types control the initiation of inflammation, which was independent of adaptive immunity. The latter delayed development of aggressive melanomas but was overridden by inflammation. Cancer Res; 70(9); 3515-25. (c)2010 AACR

Minimal tolerance to a tumor antigen encoded by a cancer-germline gene

Central tolerance toward tissue-restricted Ags is considered to rely on ectopic expression in the thymus, which was also observed for tumor Ags encoded by cancer-germline genes. It is unknown whether endogenous expression shapes the T cell repertoire against the latter Ags and explains their weak immunogenicity. We addressed this question using mouse cancer-germline gene P1A, which encodes antigenic peptide P1A(35-43) presented by H-2L(d). We made P1A-knockout (P1A-KO) mice and asked whether their anti-P1A(35-43) immune responses were stronger than those of wild-type mice and whether P1A-KO mice responded to other P1A epitopes, against which wild-type mice were tolerized. We observed that both types of mice mounted similar P1A(35-43)-specific CD8 T cell responses, although the frequency of P1A(35-43)-specific CD8 T cells generated in response to P1A-expressing tumors was slightly higher in P1A-KO mice. This higher reactivity allowed naive P1A-KO mice to reject spontaneously P1A-expressing tumors, which progressed in wild-type mice. TCR-Vβ usage of P1A(35-43)-specific CD8 cells was slightly modified in P1A-KO mice. Peptide P1A(35-43) remained the only P1A epitope recognized by CD8 T cells in both types of mice, which also displayed similar thymic selection of a transgenic TCR recognizing P1A(35-43). These results indicate the existence of a minimal tolerance to an Ag encoded by a cancer-germline gene and suggest that its endogenous expression only slightly affects diversification of the T cell repertoire against this Ag

Analysis of the TGFβ pathway in melanoma lines and tumors. A.

<p>Supernatants from Amela cell lines incubated in serum-free DMEM were either acid treated (acid) or not (no treatment) and were tested for TGFβ content using reporter line MFBF11 (see Methods). Bars represent means ± s.d. of triplicate wells in one representative experiment. Serum-free DMEM (no TGFβ) was used for baseline measurement. B. Flow cytometry analysis of GFP expression in SBE-GFP-transduced Amela and B16F10 cell lines preincubated in serum-free DMEM without addition (red lines) or in the presence of TGFβ (blue lines). Non-transduced Amela cell lines were used as control (gray filled). C. Comparasion of the expression of 3 genes associated with EMT by Amela^C and Amela^I cell lines by QRT-PCR (see text). Results are represented as fold change in relative expression where the value for Amela^C is set to 1 for each gene. Data are represented as mean ± s.e.m of two independent experiments in which 4 different lines of each type were tested. D-E. Flow cytometry analysis of GFP expression in SBE-GFP-transduced Amela^C cell lines as in (B). The effect of various inhibitors was assessed on Amela^C cells during the incubation in serum-free DMEM without (-) or with addition (+) of TGFβ. GFP expression in one Amela^C line before and after treatment with various inhibitors in the absence of TGFβ is shown in (D). In (E)results show fold change of mean GFP fluorescence intensity in Amela^C without (panel at left) or with (panel at right) addition of TGFβ. The value for Amela^I in the absence of TGFβ is set to 1. Data are represented as mean ± s.e.m of four independent experiments. ***p value < 0.001; **p value < 0.01; *p value < 0.05.</p

Genes up-regulated or down-regulated in Amela tumors involved in EMT.

(a)<p>Genes known to be involved in EMT that show higher expression in Amela versus Mela primary tumors and are expressed at similar level in Amela primary tumors and Amela lines in culture.</p>(b)<p>Ratio of gene expression as log2 primary Amela/primary Mela > 1 with p values < 0.05;</p>*<p>Ratio of gene expression as log2 primary Amela/primary Mela between 0 and 1 with p values < 0.05;</p>(c)<p>Ratio of gene expression as Log2 (primary Amela-/cultured Amela line) < 1 and/or with p value > 0.05 (ns). Log2 (primary Amela /cultured Amela line) < - 1 corresponds to higher expression in cultured Amela line than in primary Amela.</p>(d)<p>Genes known to be involved in EMT that show lower expression in primary Amela versus Mela tumors.</p

Effect of MAPK pathway inhibitors on the expression of EMT hallmark genes in Amela tumor lines.

<p>QRT-PCR analysis for expression of 6 transcripts encoding mesenchymal and epithelial markers in Amela tumor cells in the absence (value set to 1) or presence of inhibitors, as indicated. Data are represented as mean ± s.e.m of three independent experiments in which 5 samples were analyzed. ***p value < 0.001; **p value < 0.01; *p value < 0.05.</p

EMT and TGFβ pathway signatures in Amela melanomas. A.

<p>Representative gene set enrichment analysis <b>(</b>GSEA) plots of EMT (right graph) and TGFβ pathway (left graph) gene signatures. Each plot is divided into two sections. The first section (class A) shows results for gene sets that have a positive enrichment score (gene sets that show enrichment at the top of the ranked list here associated with Amela samples). The second section (class B) shows the results for gene sets that have a negative enrichment score (gene sets that show enrichment at the bottom of the ranked list here associated with Mela samples). Data provided as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049419#pone.0049419.s002" target="_blank">Fig. S2</a>. <b>B.</b> QRT-PCR analysis for expression of transcripts encoding mesenchymal and epithelial markers in Amela (white dotted bars) and Mela (black dotted bars) tumors. Values were normalized to those for skin from control mice. Data are represented as mean ± s.e.m of three independent experiments in which 7 samples of Amela and 7 samples of Mela tumors were analyzed. **p value < 0.01; *p value < 0.05.</p

Representative TGFβ Related Genes expressed in Amela tumors and cultured cell lines.

(a)<p>Genes known to be TGFβ responsive that show higher expression in Amela versus Mela primary tumors and are expressed at similar level in Amela primary tumors and in Amela lines in culture.</p>(b)<p>Ratio of gene expression as log2 primary Amela/primary Mela > 1 with p values < 0.05;</p>*<p>Ratio of gene expression as log2 primary Amela/primary Mela between 0 and 1 with p values < 0.05;</p>(c)<p>Ratio of gene expression as Log2 (primary Amela/cultured Amela line) < 1 and/or with p value > 0.05 (ns). Log2 (primary Amela/cultured Amela line) < - 1 corresponds to higher expression in cultured Amela lines than in primary tumor.</p>(d)<p>Numbered references can be found in Text S1.</p

Specific expression of Ccl2 in Amela tumors is controlled by Ras signalling pathways.

<p>(A-B) QRT-PCR analysis of Ccl2 transcript expression in induced Mela and Amela primary tumors (A) and in the corresponding cell lines in vitro (B). (C) Concentration of secreted Ccl2 in the supernatant of 24 h cultures of Amela and Mela cell lines as measured by ELISA. (D) Expression of Ccl2 transcript by QRT-PCR and (E) concentration of secreted protein by ELISA, in the absence (value set to 1) or presence of inhibitors in 24 h cultures, as indicated. (A–C) Amela tumors are represented by white dotted bars and Mela tumors by black dotted bars. For primary tumor analysis (A) 6 samples of each tumor were analyzed. For <i>in vitro</i> analysis (B), values were normalized to those for B16F10 cells. In (C), supernatants from 24 h cell cultures were tested and values are expressed as ng/ml. In D–E, culture conditions were as described in Methods. Experiments involved 10 Amela lines in (D) and 4 in (E). ****p value < 0.0001; ***p value < 0.001; **p value < 0.01; *p value < 0.05.</p

Amela tumor-intrinsic expressed genes controlling angiogenesis, invasion and cytokines.

(a)<p>Genes characterizing angiogenesis, proliferation and invasion, immune response components or chemotaxis that show higher expression in Amela versus Mela primary tumors and are expressed at similar level in Amela primary tumors and Amela lines in culture.</p>(b)<p>Ratio of gene expression as Log2 primary Amela/primary Mela > 1 with p value < 0.001;</p>(c)<p>Ratio of gene expression as Log2 primary Amela/cultured Amela line < 1 or with p value n.s.).</p