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

Phénoménologie de l'échappement tumoral dans un modèle murin de mélanome indictible

L'impact du système immunitaire dans la progression tumorale est controversé. dans les modèles de tumerus transplantées, ou spontanées exprimant des antigènes viraux, la réponse immunitaire anti-tumorale a été décrite comme soit diminuant soit favorisant la croissance tumorale. Nous avons évalué l'impact du système immunitaire sur deux tyes distincts de mélanomes induits chez la souris par la délétion conditionnelle de gènes suppresseurs de tumeur, l'exression concomitante d'un oncogène et d'un antigène tumoral. Le mélanome à croissance lente semble être "ignoré" par le système immunitaire. Alors que le mélanome "agressif" est infiltré par des cellules myéloïdes immatures et par des lymphocytes T présentant un phénotype "épuisé", en association avec une inflammation chronique systémique de type Th2. Contrairement aux autres modèles de tumeurs spontanées, le déclenchement de l'inflammation est ici indépendant de l'immunité adaptative, qui au contraire, retarde le développement tumoral, mais est supplanté par l'inflammation. Chez ces souris, des troubles dans l'hématopoïèse et un recrutement de cellules myéloïdes au niveau des organes lymphoïdes sont associés à une immunosuppression etravant la thérapie cellulaire par transfert adoptif. De façon intéressante, l'expression de gènes définissant la transition épithélio-mésenchymateuse, ainsi que des gènes codant pour des chémiokines et de cytokines immuno-modulatrices caractérisent la mélanome agressif comparé au mélanome non agressif, établissant ainsi un lien entre le processus de la transition épithélio-mésenchymateuse et des altérations des fonctions immunitairesThe role of the immune system in tumor progression is controversial. In studies of mouse transplanted tumors or spontaneous tumors expressing viral antigens, anti-tumor immune reactivity was found either to restrict or promote growth. We evaluated the impact of the immune system on two differencially aggressive melanomas developing in mice upon conditional deletion of the same tumor suppressor genes and concomitant expression of oncogene H-RasG12V and a natural cancer-germline tumor antigene. "Slow progressor" melanomas appeared to be "ignored" by the immune system. "Agressive" melanomas were infiltrated by immature myeloid cells and by T lymphocytes presenting an "exhausted" phenotype, in association with local inflammation and systemic Th2 dominant chronic inflammation. In constrast to other models, initiation of inflammation was here independent of adaptative immunity, which delayed the development of agressive melanomas, but was overridden by inflammation. In these mice, disorders in hematopoiesis and recuitment of myeloid cells to lymphoid organs were associated with immunosuppression and hampered adoptive T cell therapy. Expression of genes akin to those defining epithelial-mesenchymal transition as well as genes encoding chemokines and immuno-modulating cytokines characterized aggressive compared to slow progressor melomas, thus establishing a link between epithelial-mesenchymal transition-like processes and alterations of immune functions.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Cooperative action of CD8 T lymphocytes and natural killer cells controls tumour growth under conditions of restricted T-cell receptor diversity

In mice expressing a transgenic T-cell receptor (TCR; TCRP1A) of DBA/2 origin with reactivity towards a cancer-germline antigen P1A, the number of TCRP1A CD8+ T cells in lymphoid organs is lower in DBA/2 than in B10.D2 or B10.D2(× DBA/2)F1 mice. This reduction results from haemopoietic cell autonomous differences in the differentiation of the major histocompatibility complex class I-restricted TCRP1A thymocytes controlled by DBA/2 versus B10.D2-encoded genes. We report here that the lower number of TCRP1A CD8+ T cells in DBA/2 mice correlated with their poor resistance to P1A-expressing mastocytoma solid tumours. Functional potency of CD8+ cytolytic T lymphocytes (CTL) from the above strains was not compromised, but their number after expansion appeared to be influenced by their genetic background. Intriguingly, non-transgenic DBA/2 mice resisted P1A+ tumours more efficiently despite poor representation of P1A-specific CTL. This was partly the result of their more heterogeneous TCR repertoire, including reactivity to non-P1A tumour antigens because mice that had rejected a P1A+ tumour became resistant to a P1A− variant of the tumour. Such ‘cross-resistance’ did not develop in the TCRP1A transgenic mice. Nonetheless, reconstitution of RAGº/º mice with TCRP1A CD8+ T cells, with or without CD4+ T cells, or exclusive representation of TCRP1A CD8+ T cells in RAGº/º TCRP1A transgenic mice efficiently resisted the growth of P1A-expressing tumours. Natural killer cells present at a higher number in RAGº/º mice also contributed to tumour resistance, in part through an NKG2D-dependent mechanism. Hence, in the absence of a polyclonal T-cell repertoire, precursor frequencies of natural killer cells and tumour-specific CTL affect tumour resistance

Visualization of Cytolytic T Cell Differentiation and Granule Exocytosis with T Cells from Mice Expressing Active Fluorescent Granzyme B

<div><p>To evaluate acquisition and activation of cytolytic functions during immune responses we generated knock in (KI) mice expressing Granzyme B (GZMB) as a fusion protein with red fluorescent tdTomato (GZMB-Tom). As for GZMB in wild type (WT) lymphocytes, GZMB-Tom was absent from naïve CD8 and CD4 T cells in GZMB-Tom-KI mice. It was rapidly induced in most CD8 T cells and in a subpopulation of CD4 T cells in response to stimulation with antibodies to CD3/CD28. A fraction of splenic NK cells expressed GZMB-Tom ex vivo with most becoming positive upon culture in IL-2. GZMB-Tom was present in CTL granules and active as a protease when these degranulated into cognate target cells, as shown with target cells expressing a specific FRET reporter construct. Using T cells from mice expressing GZMB-Tom but lacking perforin, we show that the transfer of fluorescent GZMB-Tom into target cells was dependent on perforin, favoring a role for perforin in delivery of GZMB at the target cells’ plasma membranes. Time-lapse video microscopy showed Ca++ signaling in CTL upon interaction with cognate targets, followed by relocalization of GZMB-Tom-containing granules to the synaptic contact zone. A perforin-dependent step was next visualized by the fluorescence signal from the non-permeant dye TO-PRO-3 at the synaptic cleft, minutes before the labeling of the target cell nucleus, characterizing a previously undescribed synaptic event in CTL cytolysis. Transferred OVA-specific GZMB-Tom-expressing CD8 T cells acquired GZMB-Tom expression in Listeria monocytogenes-OVA infected mice as soon as 48h after infection. These GZMB-Tom positive CD8 T cells localized in the splenic T-zone where they interacted with CD11c positive dendritic cells (DC), as shown by GZMB-Tom granule redistribution to the T/DC contact zone. GZMB-Tom-KI mice thus also provide tools to visualize acquisition and activation of cytolytic function in vivo.</p></div