Molecular profiling of CD8 T cells in autochthonous melanoma identifies Maf as driver of exhaustion.

T cells infiltrating neoplasms express surface molecules typical of chronically virus-stimulated T cells, often termed "exhausted" T cells. We compared the transcriptome of "exhausted" CD8 T cells infiltrating autochthonous melanomas to those of naïve and acutely stimulated CD8 T cells. Despite strong similarities between transcriptional signatures of tumor- and virus-induced exhausted CD8 T cells, notable differences appeared. Among transcriptional regulators, Nr4a2 and Maf were highly overexpressed in tumor-exhausted T cells and significantly upregulated in CD8 T cells from human melanoma metastases. Transduction of murine tumor-specific CD8 T cells to express Maf partially reproduced the transcriptional program associated with tumor-induced exhaustion. Upon adoptive transfer, the transduced cells showed normal homeostasis but failed to accumulate in tumor-bearing hosts and developed defective anti-tumor effector responses. We further identified TGFß and IL-6 as main inducers of Maf expression in CD8 T cells and showed that Maf-deleted tumor-specific CD8 T cells were much more potent to restrain tumor growth in vivo. Therefore, the melanoma microenvironment contributes to skewing of CD8 T cell differentiation programs, in part by TGFß/IL-6-mediated induction of Maf.

Ptk7-Deficient Mice Have Decreased Hematopoietic Stem Cell Pools as a Result of Deregulated Proliferation and Migration.

Hematopoietic stem cells (HSCs) located in adult bone marrow or fetal liver in mammals produce all cells from the blood system. At the top of the hierarchy are long-term HSCs endowed with lifelong self-renewal and differentiation properties. These features are controlled through key microenvironmental cues and regulatory pathways, such as Wnt signaling. We showed previously that PTK7, a tyrosine kinase receptor involved in planar cell polarity, plays a role in epithelial Wnt signaling; however, its function in hematopoiesis has remained unexplored. In this article, we show that PTK7 is expressed by hematopoietic stem and progenitor cells, with the highest level of protein expression found on HSCs. Taking advantage of a Ptk7-deficient mouse strain, we demonstrate that loss of Ptk7 leads to a diminished pool of HSCs but does not affect in vitro or in vivo hematopoietic cell differentiation. This is correlated with increased quiescence and reduced homing abilities of Ptk7-deficient hematopoietic stem and progenitor cells, unraveling novel and unexpected functions for planar cell polarity pathways in HSC fate.

The tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20) imposes a brake on antitumor activity of CD8 T cells.

The transcription factor NF-κB is central to inflammatory signaling and activation of innate and adaptive immune responses. Activation of the NF-κB pathway is tightly controlled by several negative feedback mechanisms, including A20, an ubiquitin-modifying enzyme encoded by the tnfaip3 gene. Mice with selective deletion of A20 in myeloid, dendritic, or B cells recapitulate some human inflammatory pathology. As we observed high expression of A20 transcripts in dysfunctional CD8 T cells in an autochthonous melanoma, we analyzed the role of A20 in regulation of CD8 T-cell functions, using mice in which A20 was selectively deleted in mature conventional T cells. These mice developed lymphadenopathy and some organ infiltration by T cells but no splenomegaly and no detectable pathology. A20-deleted CD8 T cells had increased sensitivity to antigen stimulation with production of large amounts of IL-2 and IFNγ, correlated with sustained nuclear expression of NF-κB components reticuloendotheliosis oncogene c-Rel and p65. Overexpression of A20 by retroviral transduction of CD8 T cells dampened their intratumor accumulation and antitumor activity. In contrast, relief from the A20 brake in NF-κB activation in adoptively transferred antitumor CD8 T cells led to improved control of melanoma growth. Tumor-infiltrating A20-deleted CD8 T cells had enhanced production of IFNγ and TNFα and reduced expression of the inhibitory receptor programmed cell death 1. As manipulation of A20 expression in CD8 T cells did not result in pathologic manifestations in the mice, we propose it as a candidate to be targeted to increase antitumor efficiency of adoptive T-cell immunotherapy.

The PTK7 and ROR2 Protein Receptors Interact in the Vertebrate WNT/Planar Cell Polarity (PCP) Pathway.

The non-canonical WNT/planar cell polarity (WNT/PCP) pathway plays important roles in morphogenetic processes in vertebrates. Among WNT/PCP components, protein tyrosine kinase 7 (PTK7) is a tyrosine kinase receptor with poorly defined functions lacking catalytic activity. Here we show that PTK7 associates with receptor tyrosine kinase-like orphan receptor 2 (ROR2) to form a heterodimeric complex in mammalian cells. We demonstrate that PTK7 and ROR2 physically and functionally interact with the non-canonical WNT5A ligand, leading to JNK activation and cell movements. In the Xenopus embryo, Ptk7 functionally interacts with Ror2 to regulate protocadherin papc expression and morphogenesis. Furthermore, we show that Ptk7 is required for papc activation induced by Wnt5a. Interestingly, we find that Wnt5a stimulates the release of the tagged Ptk7 intracellular domain, which can translocate into the nucleus and activate papc expression. This study reveals novel molecular mechanisms of action of PTK7 in non-canonical WNT/PCP signaling that may promote cell and tissue movements.

Netrin 1 contributes to vascular remodeling in the subventricular zone and promotes progenitor emigration after demyelination.

Neural stem cells are maintained in the adult brain, sustaining structural and functional plasticity and to some extent participating in brain repair. A thorough understanding of the mechanisms and factors involved in endogenous stem/progenitor cell mobilization is a major challenge in the promotion of spontaneous brain repair. The main neural stem cell niche in the adult brain is the subventricular zone (SVZ). Following demyelination insults, SVZ-derived progenitors act in concert with oligodendrocyte precursors to repopulate the lesion and replace lost oligodendrocytes. Here, we showed robust vascular reactivity within the SVZ after focal demyelination of the corpus callosum in adult mice, together with a remarkable physical association between these vessels and neural progenitors exiting from their niche. Endogenous progenitor cell recruitment towards the lesion was significantly reduced by inhibiting post-lesional angiogenesis in the SVZ using anti-VEGF blocking antibody injections, suggesting a facilitating role of blood vessels for progenitor cell migration towards the lesion. We identified netrin 1 (NTN1) as a key factor upregulated within the SVZ after demyelination and involved in local angiogenesis and progenitor cell migration. Blocking NTN1 expression using a neutralizing antibody inhibited both lesion-induced vascular reactivity and progenitor cell recruitment at the lesion site. We propose a model in which SVZ progenitors respond to a demyelination lesion by NTN1 secretion that both directly promotes cell emigration and contributes to local angiogenesis, which in turn indirectly facilitates progenitor cell emigration from the niche.

Control of CD8 T cell proliferation and terminal differentiation by active STAT5 and CDKN2A/CDKN2B.

CD8 T cells used in adoptive immunotherapy may be manipulated to optimize their effector functions, tissue-migratory properties and long-term replicative potential. We reported that antigen-stimulated CD8 T cells transduced to express an active form of the transcription factor signal transducer and activator of transcription 5 (STAT5CA) maintained these properties upon adoptive transfer. We now report on the requirements of STAT5CA-expressing CD8 T cells for cell survival and proliferation in vivo. We show that STAT5CA expression allows for greater expansion of T cells in vivo, while preserving dependency on T-cell-receptor-mediated tonic stimulation for their in vivo maintenance and return to a quiescent stage. STAT5CA expression promotes the formation of a large pool of effector memory T cells that respond upon re-exposure to antigen and present an increased sensitivity to γc receptor cytokine engagement for STAT5 phosphorylation. In addition, STAT5CA expression prolongs the survival of what would otherwise be short-lived terminally differentiated KLRG1-positive effector cells with up-regulated expression of the senescence-associated p16(INK) (4A) transcripts. However, development of a KLRG1-positive CD8 T cell population was independent of either p16(INK) (4A) or p19(ARF) expression (as shown using T cells from CDKN2A(-/-) mice) but was associated with expression of transcripts encoding p15(INK) (4B) , another protein involved in senescence induction. We conclude that T-cell-receptor- and cytokine-dependent regulation of effector T cell homeostasis, as well as mechanisms leading to senescent features of a population of CD8 T cells are maintained in STAT5CA-expressing CD8 T cells, even for cells that are genetically deficient in expression of the tumour suppressors p16(INK) (4A) and p19(ARF) .