Madiha Derouazi

Immune infiltration of spontaneous mouse astrocytomas is dominated by immunosuppressive cells from early stages of tumor development

Immune infiltration of advanced human gliomas has been shown, but it is doubtful whether these immune cells affect tumor progression. It could be hypothesized that this infiltrate reflects recently recruited immune cells that are immediately overwhelmed by a high tumor burden. Alternatively, if there is earlier immune detection and infiltration of the tumor, the question arises as to when antitumor competency is lost. To address these issues, we analyzed a transgenic mouse model of spontaneous astrocytoma (GFAP-V(12)HA-ras mice), which allows the study of immune interactions with developing glioma, even at early asymptomatic stages. T cells, including a significant proportion of Tregs, are already present in the brain before symptoms develop, followed later by macrophages, natural killer cells, and dendritic cells. The effector potential of CD8 T-cells is defective, with the absence of granzyme B expression and low expression of IFN-gamma, tumor necrosis factor, and interleukin 2. Overall, our results show an early defective endogenous immune response to gliomas, and local accumulation of immunosuppressive cells at the tumor site. Thus, the antiglioma response is not simply overwhelmed at advanced stages of tumor growth, but is counterbalanced by an inhibitory microenvironment from the outset. Nevertheless, we determined that effector molecule expression (granzyme B, IFN-gamma) by brain-infiltrating CD8 T-cells could be enhanced, despite this unfavorable milieu, by strong immune stimuli. This potential to modulate the strong imbalance in local antiglioma immunity is encouraging for the development and optimization of future glioma immunotherapies.

Novel Cell-Penetrating Peptide-Based Vaccine Induces Robust CD4+ and CD8+ T Cell–Mediated Antitumor Immunity

Vaccines that can coordinately induce multi-epitope T cell-mediated immunity, T helper functions, and immunologic memory may offer effective tools for cancer immunotherapy. Here, we report the development of a new class of recombinant protein cancer vaccines that deliver different CD8(+) and CD4(+) T-cell epitopes presented by MHC class I and class II alleles, respectively. In these vaccines, the recombinant protein is fused with Z12, a novel cell-penetrating peptide that promotes efficient protein loading into the antigen-processing machinery of dendritic cells. Z12 elicited an integrated and multi-epitopic immune response with persistent effector T cells. Therapy with Z12-formulated vaccines prolonged survival in three robust tumor models, with the longest survival in an orthotopic model of aggressive brain cancer. Analysis of the tumor sites showed antigen-specific T-cell accumulation with favorable modulation of the balance of the immune infiltrate. Taken together, the results offered a preclinical proof of concept for the use of Z12-formulated vaccines as a versatile platform for the development of effective cancer vaccines.

Phenotypic switch of CD8+ T cells reactivated under hypoxia toward IL-10 secreting, poorly proliferative effector cells

CD8+ T cells controlling pathogens or tumors must function at sites where oxygen tension is frequently low, and never as high as under atmospheric culture conditions. However, T‐cell function in vivo is generally analyzed indirectly, or is extrapolated from in vitro studies under nonphysiologic oxygen tensions. In this study, we delineate the role of physiologic and pathologic oxygen tension in vitro during reactivation and differentiation of tumor‐specific CD8+ T cells. Using CD8+ T cells from pmel‐1 mice, we observed that the generation of CTLs under 5% O2, which corresponds to physioxia in lymph nodes, gave rise to a higher effector signature than those generated under atmospheric oxygen fractions (21% O2). Hypoxia (1% O2) did not modify cytotoxicity, but decreasing O2 tensions during CTL and CD8+ tumor‐infiltrating lymphocyte reactivation dose‐dependently decreased proliferation, induced secretion of the immunosuppressive cytokine IL‐10, and upregulated the expression of CD137 (4‐1BB) and CD25. Overall, our data indicate that oxygen tension is a key regulator of CD8+ T‐cell function and fate and suggest that IL‐10 release may be an unanticipated component of CD8+ T cell‐mediated immune responses in most in vivo microenvironments.

Synergy between CD8 T Cells and Th1 or Th2 Polarised CD4 T Cells for Adoptive Immunotherapy of Brain Tumours

The feasibility of cancer immunotherapy mediated by T lymphocytes is now a clinical reality. Indeed, many tumour associated antigens have been identified for cytotoxic CD8 T cells, which are believed to be key mediators of tumour rejection. However, for aggressive malignancies in specialised anatomic sites such as the brain, a limiting factor is suboptimal tumour infiltration by CD8 T cells. Here we take advantage of recent advances in T cell biology to differentially polarise CD4 T cells in order to explore their capacity to enhance immunotherapy. We used an adoptive cell therapy approach to work with clonal T cell populations of defined specificity. Th1 CD4 T cells preferentially homed to and accumulated within intracranial tumours compared with Th2 CD4 T cells. Moreover, tumour-antigen specific Th1 CD4 T cells enhanced CD8 T cell recruitment and function within the brain tumour bed. Survival of mice bearing intracranial tumours was significantly prolonged when CD4 and CD8 T cells were co-transferred. These results should encourage further definition of tumour antigens recognised by CD4 T cells, and exploitation of both CD4 and CD8 T cell subsets to optimise T cell therapy of cancer.

Optimization of a Type III Secretion System-Based Pseudomonas aeruginosa Live Vector for Antigen Delivery

ABSTRACT During the last few years, the use of type III secretion system-based bacterial vectors for immunotherapy purposes has been assessed in various applications. We showed that a type III secretion-based Pseudomonas aeruginosa vector delivering the ovalbumin (OVA) antigen induced an efficient specific CD8+ T-lymphocyte immune response against OVA-expressing cells. Because of the intrinsic toxicity of the vector, further virulence attenuation was needed. Therefore, we explored the effects of the deletion of quorum-sensing genes and the aroA gene toward toxicity and efficiency of the vector strain. The aroA mutation in our strain (making the strain auxotrophic for aromatic amino acids) conferred a strikingly reduced toxicity, with the bacterial lethal dose being more than 100 times higher than that of the parental strain. The quorum-sensing gene mutation alone was associated with a slightly reduced toxicity. In a prophylactic OVA-expressing melanoma mouse model, an OVA-delivering aroA-deficient mutant was the most efficient at a low dose (105), but dose enhancement was not associated with a greater immune response. The quorum-sensing-deficient strain was the most efficient at a mild dose (106), but this dose was close to the toxic dose. Combination of both mutations conferred the highest efficiency at an elevated dose (107), in agreement with the known negative effects of quorum-sensing molecules upon T-cell activation. In conclusion, we have obtained a promising immunotherapy vector regarding toxicity and efficiency for further developments in both antitumor and anti-infectious strategies.

Optimal epitope composition after antigen screening using a live bacterial delivery vector: application to TRP-2.

Immunotherapeutic approaches, based on the generation of tumor-specific cytotoxic T-lymphocytes (CTL), are currently emerging as promising strategies of anti-tumor therapy. The potential use of attenuated bacteria as engineered vectors for vaccine development offers several advantages, including the stimulation of innate immunity. We developed an attenuated live bacterial vector using the type III secretion system (TTSS) of Pseudomonas aeruginosa to deliver in vivo tumor antigens. Using an inducible and rapid expression plasmid, vaccination with several antigens of different length and epitope composition, including TRP-2, gp100 and MUC18, was evaluated against glioma tumor cells. We observed similar CTL immunity and T-cell receptor (TCR) repertoire diversity with the vaccines, TRP2125-243, TRP2L125-376 and TRP2S291-376. However, only immunization with TRP2L125-376 induced significant anti-tumor immunity. Taken together, our data indicate the importance of the epitopes composition and/or peptide length of these peptides for inducing cytotoxic T-lymphocyte (CTL) mediated immunity. Characteristics that consistently improved anti-tumor immunity include: long peptides with immunodominant and cryptic CD8+ epitopes, and strong CD4+ Th epitopes. Our bacterial vector is versatile, easy-to-use and quick to produce. This vector is suitable for rapide screening and evaluation of antigens of varying length and epitope composition.

ER–mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem‐like cells

Glioblastoma is a highly heterogeneous aggressive primary brain tumor, with the glioma stem‐like cells (GSC) being more sensitive to cytotoxic lymphocyte‐mediated killing than glioma differentiated cells (GDC). However, the mechanism behind this higher sensitivity is unclear. Here, we found that the mitochondrial morphology of GSCs modulates the ER–mitochondria contacts that regulate the surface expression of sialylated glycans and their recognition by cytotoxic T lymphocytes and natural killer cells. GSCs displayed diminished ER–mitochondria contacts compared to GDCs. Forced ER–mitochondria contacts in GSCs increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. Therefore, mitochondrial morphology and dynamism dictate the ER–mitochondria contacts in order to regulate the surface expression of certain glycans and thus play a role in GSC recognition and elimination by immune effector cells. Targeting the mitochondrial morphology, dynamism, and contacts with the ER could be an innovative strategy to deplete the cancer stem cell compartment to successfully treat glioblastoma.

Enhancing Antitumor Immune Responses by Optimized Combinations of Cell-penetrating Peptide-based Vaccines and Adjuvants

Cell penetrating peptides (CPPs) from the protein ZEBRA are promising candidates to exploit in therapeutic cancer vaccines, since they can transport antigenic cargos into dendritic cells and induce tumor-specific T cells. Employing CPPs for a given cancer indication will require engineering to include relevant tumor-associated epitopes, administration with an appropriate adjuvant, and testing for antitumor immunity. We assessed the importance of structural characteristics, efficiency of in vitro transduction of target cells, and choice of adjuvant in inducing the two key elements in antitumor immunity, CD4 and CD8 T cells, as well as control of tumor growth in vivo. Structural characteristics associated with CPP function varied according to CPP truncations and cargo epitope composition, and correlated with in vitro transduction efficiency. However, subsequent in vivo capacity to induce CD4 and CD8 T cells was not always predicted by in vitro results. We determined that the critical parameter for in vivo efficacy using aggressive mouse tumor models was the choice of adjuvant. Optimal pairing of a particular ZEBRA-CPP sequence and antigenic cargo together with adjuvant induced potent antitumor immunity. Our results highlight the irreplaceable role of in vivo testing of novel vaccine constructs together with adjuvants to select combinations for further development.

Mechanistic insights into the efficacy of cell penetrating peptide-based cancer vaccines

Immunotherapies are increasingly used to treat cancer, with some outstanding results. Immunotherapy modalities include therapeutic vaccination to eliminate cancer cells through the activation of patient’s immune system against tumor-derived antigens. Nevertheless, the full potential of therapeutic vaccination has yet to be demonstrated clinically because many early generation vaccines elicited low-level immune responses targeting only few tumor antigens. Cell penetrating peptides (CPPs) are highly promising tools to advance the field towards clinical success. CPPs efficiently penetrate cell membranes, even when linked to antigenic cargos, which can induce both CD8 and CD4 T-cell responses. Pre-clinical studies demonstrated that targeting multiple tumor antigens, even those considered to be poorly immunogenic, led to tumor regression. Therefore, CPP-based cancer vaccines represent a flexible and powerful means to extend therapeutic vaccination to many cancer indications. Here, we review recent findings in CPP development and discuss their use in next generation immunotherapies.

Identification of a novel population of highly cytotoxic c‐Met‐expressing CD8+ T lymphocytes

CD8+ cytotoxic T lymphocytes (CTLs) are critical mediators of anti‐tumor immunity, and controlling the mechanisms that govern CTL functions could be crucial for enhancing patient outcome. Previously, we reported that hepatocyte growth factor (HGF) limits effective murine CTL responses via antigen‐presenting cells. Here, we show that a fraction of murine effector CTLs expresses the HGF receptor c‐Met (c‐Met+ CTLs). Phenotypic and functional analysis of c‐Met+ CTLs reveals that they display enhanced cytolytic capacities compared to their c‐Met− CTL counterparts. Furthermore, HGF directly restrains the cytolytic function of c‐Met+ CTLs in cell‐mediated cytotoxicity reactions in vitro and in vivo and abrogates T‐cell responses against metastatic melanoma in vivo. Finally, we establish in three murine tumor settings and in human melanoma tissues that c‐Met+ CTLs are a naturally occurring CD8+ T‐cell population. Together, our findings suggest that the HGF/c‐Met pathway could be exploited to control CD8+ T‐cell‐mediated anti‐tumor immunity.