Δ133p53α enhances metabolic and cellular fitness of TCR-engineered T cells and promotes superior antitumor immunity

Background Tumor microenvironment-associated T cell senescence is a key limiting factor for durable effective cancer immunotherapy. A few studies have demonstrated the critical role of the tumor suppressor TP53-derived p53 isoforms in cellular senescence process of non-immune cells. However, their role in lymphocytes, in particular tumor-antigen (TA) specific T cells remain largely unexplored.Methods Human T cells from peripheral blood were retrovirally engineered to coexpress a TA-specific T cell receptor and the Δ133p53α-isoform, and characterized for their cellular phenotype, metabolic profile and effector functions.Results Phenotypic analysis of Δ133p53α-modified T cells revealed a marked reduction of the T-cell inhibitory molecules (ie, CD160 and TIGIT), a lower frequency of senescent-like CD57+ and CD160+ CD8+ T cell populations, and an increased number of less differentiated CD28+ T cells. Consistently, we demonstrated changes in the cellular metabolic program toward a quiescent T cell state. On a functional level, Δ133p53α-expressing T cells acquired a long-term proliferative capacity, showed superior cytokine secretion and enhanced tumor-specific killing in vitro and in mouse tumor model. Finally, we demonstrated the capacity of Δ133p53α to restore the antitumor response of senescent T cells isolated from multiple myeloma patients.Conclusion This study uncovered a broad effect of Δ133p53α isoform in regulating T lymphocyte function. Enhancing fitness and effector functions of senescent T cells by modulation of p53 isoforms could be exploited for future translational research to improve cancer immunotherapy and immunosenescence-related diseases

A Concerted HIF-1α/MT1-MMP Signalling Axis Regulates the Expression of the 3BP2 Adaptor Protein in Hypoxic Mesenchymal Stromal Cells

Increased plasticity, migratory and immunosuppressive abilities characterize mesenchymal stromal cells (MSC) which enable them to be active participants in the development of hypoxic solid tumours. Our understanding of the oncogenic adaptation of MSC to hypoxia however lacks the identification and characterization of specific biomarkers. In this study, we assessed the hypoxic regulation of 3BP2/SH3BP2 (Abl SH3-binding protein 2), an immune response adaptor/scaffold protein which regulates leukocyte differentiation and motility. Gene silencing of 3BP2 abrogated MSC migration in response to hypoxic cues and generation of MSC stably expressing the transcription factor hypoxia inducible factor 1alpha (HIF-1α) resulted in increased endogenous 3BP2 expression as well as cell migration. Analysis of the 3BP2 promoter sequence revealed only one potential HIF-1α binding site within the human but none in the murine sequence. An alternate early signalling cascade that regulated 3BP2 expression was found to involve membrane type-1 matrix metalloproteinase (MT1-MMP) transcriptional regulation which gene silencing abrogated 3BP2 expression in response to hypoxia. Collectively, we provide evidence for a concerted HIF-1α/MT1-MMP signalling axis that explains the induction of adaptor protein 3BP2 and which may link protein binding partners together and stimulate oncogenic MSC migration. These mechanistic observations support the potential for malignant transformation of MSC within hypoxic tumour stroma and may contribute to evasion of the immune system by a tumour

Fonctions de la molécule d'adhésion MCAM/CD146 dans l'interaction leucocyte-endothélium

Bien que la molécule d adhésion MCAM/CD146 (Melanoma Cell Adhesion Molecule) soit un marqueur des progéniteurs hématopoïétiques, des lymphocytes T activés et des endothéliums, sa fonction au niveau des ces tissus n est pas encore élucidée. MCAM/CD146 est une glycoprotéine du sous-groupe V-V-C2-C2-C2 de la superfamille des Immunoglobulines. Elle existe sous deux isoformes différentes suivant la taille de la région cytoplasmique : MCAM-l (long) et MCAM-s (court). Le but de cette étude était de déterminer la fonction de la molécule MCAM dans les différentes étapes du recrutement leucocytaire par l endothélium in vitro. Nous avons utilisé d une part, une lignée de lymphocytes activés CD4+MCAM-l+ (CETM4) et d autre part, la lignée Natural Killer (NKL1) exprimant les différentes isoformes de la molécule MCAM. Dans un système de flux continu in vitro, l expression de l isoforme MCAM-l, diminue la vitesse du roulement et augmente l adhésion de NKL1 et CEMT4 sur la monocouche de cellules endothéliales. L utilisation d anticorps spécifiques et de protéines MCAM solubles lors de ces expériences confirme l implication de l isoforme MCAM-l dans ces processus. Par ailleurs, l expression de MCAM-l dans les NKL1 induit l augmentation du nombre et de la taille des microvillosités et favorise les interactions moléculaires lors du rolling . Enfin, les cellules MDCK ont permis d établir un adressage différentiel des isoformes de MCAM dans un épithélium polarisé : MCAM-l est localisé au pôle baso-latéral alors que MCAM-s est détectée par défaut au pôle apical suggérant des rôles différents. L ensemble de ces résultats permet l établissement d un modèle présentant les rôles potentiels de cette molécule dans l interaction leucocyte-endothélium.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Stable expression of HIF-1α increases 3BP2 basal expression and MSC migration.

<p>MSC stably expressing a ΔODD HIF-1α mutant (MSC-HIF) were generated as described in the Methods section. (A) Basal migration of MSC and MSC-HIF was performed as described in the Methods section. Values of cell migration are means of three independent experiments (*<i>p</i><0.05 versus normoxic MSC). (B) Cell lysates were isolated, western blotting and immunodetection was performed with anti-3BP2 and anti-GAPDH antibodies as described in the Methods section. (C) Total RNA was extracted, and qRT-PCR was used to assess 3BP2 gene expression. (D) Cell migration was quantified as described in the Methods section in Mock- (white bars) and si3BP2- (black bars) transfected MSC or MSC-HIF-1α. Values of cell migration are means of three independent experiments (*<i>p</i><0.05 versus normoxic mock MSC or MSC-HIF); Bars, ±SD.</p

3BP2 induction requires MT1-MMP cytoplasmic domain-mediated signalling.

<p>(A) MSC were transiently transfected with pcDNA3.1 (Mock, white bars), a plasmid encoding full length MT1-MMP (Wt, black bars), or a plasmid encoding for a cytoplasmic domain-deleted form of MT1-MMP (Δ-cyto, grey bars). Cells were then cultured under normoxic culture conditions, total RNA was extracted, and qRT-PCR was used to assess 3BP2 gene expression. (B) Gelatin zymography of the conditioned media was used to demonstrate efficient cell surface targeting of the respective Wt and Δ-cyto MT1-MMP recombinant proteins and subsequent ability to retain extracellular catalytic functions and to activate the secreted latent proMMP-2 into active MMP-2. (C) Cell lysates were isolated, western blotting and immunodetection was performed with antibodies recognizing the MT1-MMP catalytic or cytoplasmic domain, anti-3BP2 and anti-GAPDH as described in the Methods section. Values are means of two independent experiments, each performed in triplicates (*<i>p</i><0.05 versus Wt-MT1-MMP control); Bars, ±SD.</p

Wnt3a activates dormant c-Kit(-) bone marrow-derived cells with short-term multilineage hematopoietic reconstitution capacity.

Quiescent cells lacking expression of mature lineage makers and the c-Kit receptor reside in adult bone marrow. Despite their phenotypic similarity to hematopoietic stem cells, these Lin(-)Sca-1(+)c-Kit(-) cells lack myeloid and erythroid potential and long-term hematopoietic repopulating capacity, whereas, recent studies have functionally demonstrated that the Lin(-)Sca-1(+)c-Kit(-) population contains early lymphoid-committed progenitors. Examining the role of Wnt signaling in regulation of this population, we found that c-Kit(-) cells express diverse Wnt receptors and proliferate upon Wnt pathway activation in vitro and in vivo. Stimulation with Wnt3a, but not Wnt5a or Wnt11, promoted c-Kit(-) cells to give rise to myeloid and erythroid progenitors with robust self-renewal capacity measured by clonal replating. In addition, Wnt3a-stimulated c-Kit(-) cells gave rise to all hematopoietic lineages (lymphoid, myeloid, and erythroid) upon transplant into the liver of newborn recipient mice. Our study reveals that Wnt3a activates unique cell fate decisions of dormant c-Kit(-) that promotes short-term multilineage reconstitution capacity in vivo, thereby revealing a unique role for Wnt activation in hematopoiesis. Overall, our results highlight the potential of utilizing signaling molecules known to have instructive roles in regeneration to discover cell subsets residing in adult organisms with unexploited regenerative capacity

3BP2 silencing abrogates MSC migration in response to hypoxic culture conditions.

<p>MSC were transiently transfected with scrambled sequences or with 3BP2 siRNA as described in the Methods section. (A) Immunoblotting of 3BP2 protein expression was evaluated in cell lysates from Mock (white bars) and siRNA (black bars) experiments. Total RNA was extracted, and qRT-PCR was used to assess 3BP2 gene expression knockdown efficiency. Apoptotic cell death was assessed by the fluorometric caspase-3 activity as described in the Methods section. (B) Cells were harvested and seeded onto Boyden chambers using gelatin-coated filters to assess cell migration under normoxic or hypoxic culture conditions. (C) Cell migration was quantified as described in the Methods section and expressed as x-fold induction over Mock-transfected cells. Values of cell migration are means of three independent experiments (*<i>p</i><0.05 versus normoxic mock); Bars, ±SD.</p

Gene silencing of HIF-1α antagonizes the effects of hypoxia on 3BP2 gene and protein expression.

<p>MSC were transiently transfected with scrambled sequences (Mock, white bars, open circles) or HIF-1α siRNA (black bars, closed circles) as described in the Methods section. Cells were then cultured under normoxic or hypoxic culture conditions as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021511#pone-0021511-g001" target="_blank">Fig. 1</a>. (A) Apoptotic cell death was assessed using the fluorometric caspase-3 activity assay as described in the Methods section. Concanavalin-A-treated MSC (grey bars) was used as a positive inducer of caspase-3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021511#pone.0021511-Annabi3" target="_blank">[16]</a>. Total RNA was extracted, and qRT PCR was used to assess 3BP2 and HIF-1α transcript levels. (B) 3BP2 gene expression was assessed by qRT-PCR in Mock-transfected and in siHIF-1α-transfected cells that were subsequently cultured under hypoxic conditions. (C) Mesenchymal stromal cells were transiently transfected with scrambled sequences or HIF-1α siRNA as described in the Methods section. Cells were then cultured under normoxic or hypoxic culture conditions, cell lysates were isolated, western blotting and immunodetection was performed with anti-3BP2 and anti-GAPDH antibodies. A representative blot is shown out of two. (D) Scanning densitometry was used to assess protein expression described in panel C, and the ratio of 3BP2/GAPDH expression was represented. Values in (A) and (B) are means of two independent experiments, each performed in triplicates (*<i>p</i><0.05 versus mock control in (A) or mock at time = 0 hr hypoxia in (B)); Bars, ±SD.</p

Dual Role of Melanoma Cell Adhesion Molecule (MCAM)/CD146 in Lymphocyte Endothelium Interaction: MCAM/CD146 Promotes Rolling via Microvilli Induction in Lymphocyte and Is an Endothelial Adhesion Receptor

The melanoma cell adhesion molecule (MCAM)/CD146 is expressed as two isoforms differing by their cytoplasmic domain (MCAM long (MCAM-l) and MCAM short (MCAM-s)). MCAM being expressed by endothelial cells and activated T cells, we analyzed its involvement in lymphocyte trafficking. The NK cell line NKL1 was transfected by MCAM isoforms and submitted to adhesion on both the endothelial cell monolayer and recombinant molecules under shear stress. MCAM-l transfection reduced rolling velocity and increased NKL1 adhesion on the endothelial cell monolayer and VCAM-1. Scanning electron microscopy revealed that MCAM-l induced microvilli formation and extension. In contrast, MCAM short or mock transfection had no effect on adhesion of NKL1 cells and microvilli formation. As shown by mutagenesis, serine 32 of the MCAM-l cytoplasmic tail, belonging to a putative protein kinase C phosphorylation site, was necessary for MCAM-l-actin cytoskeleton interaction and microvilli induction. Accordingly, chelerythrine chloride, a protein kinase C inhibitor, abolished MCAM-l-induced microvilli and rolling of MCAM-l-transfected NKL1 cells. Inhibition of adhesion under shear stress by anti-MCAM Abs suggested that both lymphoid MCAM-l and endothelial MCAM were also directly involved in lymphocyte endothelium interaction. MCAM-l-transfected NKL1 and activated CD4 T cells adhered to rMCAM under shear stress whereas anti-MCAM Ab treatment inhibited this process. Taken together, these data establish that MCAM is involved in the initial steps of lymphocyte endothelium interaction. By promoting the rolling on the inflammation marker VCAM-1 via microvilli induction and displaying adhesion receptor activity involving possible homophilic MCAM-l-MCAM-l interactions, MCAM might be involved in the recruitment of activated T cells to inflammation sites