Alessio Lanna

p38 signaling inhibits mTORC1-independent autophagy in senescent human CD8+ T cells

T cell senescence is thought to contribute to immune function decline, but the pathways that mediate senescence in these cells are not clear. Here, we evaluated T cell populations from healthy volunteers and determined that human CD8+ effector memory T cells that reexpress the naive T cell marker CD45RA have many characteristics of cellular senescence, including decreased proliferation, defective mitochondrial function, and elevated levels of both ROS and p38 MAPK. Despite their apparent senescent state, we determined that these cells secreted high levels of both TNF-α and IFN-γ and showed potent cytotoxic activity. We found that the senescent CD45RA-expressing population engaged anaerobic glycolysis to generate energy for effector functions. Furthermore, inhibition of p38 MAPK signaling in senescent CD8+ T cells increased their proliferation, telomerase activity, mitochondrial biogenesis, and fitness; however, the extra energy required for these processes did not arise from increased glucose uptake or oxidative phosphorylation. Instead, p38 MAPK blockade in these senescent cells induced an increase in autophagy through enhanced interactions between p38 interacting protein (p38IP) and autophagy protein 9 (ATG9) in an mTOR-independent manner. Together, our findings describe fundamental metabolic requirements of senescent primary human CD8+ T cells and demonstrate that p38 MAPK blockade reverses senescence via an mTOR-independent pathway.

The kinase p38 activated by the metabolic regulator AMPK and scaffold TAB1 drives the senescence of human T cells

In T lymphocytes, p38 MAP kinase (MAPK) regulates pleiotropic functions and is activated by canonical MAPK signaling or the alternative T cell receptor (TCR) activation pathway. Here we show that senescent human T cells lack the canonical and alternative pathways of p38 activation, but spontaneously engage the metabolic master regulator AMPK to trigger p38 recruitment to the scaffold TAB1 causing p38 auto-phosphorylation. Signaling via this pathway inhibits telomerase activity, T cell proliferation and expression of key components of the TCR signalosome. Our findings identify an unrecognized mode of p38 activation in T cells driven by intracellular changes such as low-nutrient and DNA-damage signaling (‘intra-sensory’ pathway). The proliferative defect of senescent T cells is reversed by blocking AMPK-TAB1-dependent p38 activation.In T lymphocytes, the mitogen-activated protein kinase (MAPK) p38 regulates pleiotropic functions and is activated by canonical MAPK signaling or the alternative activation pathway downstream of the T cell antigen receptor (TCR). Here we found that senescent human T cells lacked the canonical and alternative pathways for the activation of p38 but spontaneously engaged the metabolic master regulator AMPK to trigger recruitment of p38 to the scaffold protein TAB1, which caused autophosphorylation of p38. Signaling via this pathway inhibited telomerase activity, T cell proliferation and the expression of key components of the TCR signalosome. Our findings identify a previously unrecognized mode for the activation of p38 in T cells driven by intracellular changes such as low-nutrient and DNA-damage signaling (an intrasensory pathway). The proliferative defect of senescent T cells was reversed by blockade of AMPK-TAB1–dependent activation of p38.

Retroviral and Lentiviral Vectors for the Induction of Immunological Tolerance

Retroviral and lentiviral vectors have proven to be particularly efficient systems to deliver genes of interest into target cells, either in vivo or in cell cultures. They have been used for some time for gene therapy and the development of gene vaccines. Recently retroviral and lentiviral vectors have been used to generate tolerogenic dendritic cells, key professional antigen presenting cells that regulate immune responses. Thus, three main approaches have been undertaken to induce immunological tolerance; delivery of potent immunosuppressive cytokines and other molecules, modification of intracellular signalling pathways in dendritic cells, and de-targeting transgene expression from dendritic cells using microRNA technology. In this review we briefly describe retroviral and lentiviral vector biology, and their application to induce immunological tolerance.

Senescence of T Lymphocytes: Implications for Enhancing Human Immunity

As humans live longer, a central concern is to find ways to maintain their health as they age. Immunity declines during ageing, as shown by the increased susceptibility to infection by both previously encountered and new pathogens and by the decreased efficacy of vaccination. It is therefore crucial to understand the mechanisms responsible for this decrease in immunity and to develop new strategies to enhance immune function in older humans. We discuss here how the induction of senescence alters leukocyte, and specifically T cell, function. An emerging concept is that senescence and nutrient sensing-signalling pathways within T cells converge to regulate functional responses, and the manipulation of these pathways may offer new ways to enhance immunity during ageing.

MODULATING CO-STIMULATION DURING ANTIGEN PRESENTATION TO ENHANCE CANCER IMMUNOTHERAPY.

One of the key roles of the immune system is the identification of potentially dangerous pathogens or tumour cells, and raising a wide range of mechanisms to eliminate them from the organism. One of these mechanisms is activation and expansion of antigen-specific cytotoxic T cells, after recognition of antigenic peptides on the surface of antigen presenting cells such as dendritic cells (DCs). However, DCs also process and present autoantigens. Therefore, antigen presentation has to occur in the appropriate context to either trigger immune responses or establishing immunological tolerance. This is achieved by co-stimulation of T cells during antigen presentation. Co-stimulation consists on the simultaneous binding of ligand-receptor molecules at the immunological synapse which will determine the type and extent of T cell responses. In addition, the type of cytokines/chemokines present during antigen presentation will influence the polarisation of T cell responses, whether they lead to tolerance, antibody responses or cytotoxicity. In this review, we will focus on approaches manipulating co-stimulation during antigen presentation, and the role of cytokine stimulation on effective T cell responses. More specifically, we will address the experimental strategies to interfere with negative co-stimulation such as that mediated by PD-L1 (Programmed cell death 1 ligand 1)/PD-1 (Programmed death 1) to enhance anti-tumour immunity.

Anti-melanoma vaccines engineered to simultaneously modulate cytokine priming and silence PD-L1 characterized using ex vivo myeloid-derived suppressor cells as a readout of therapeutic efficacy

Efficacious antitumor vaccines strongly stimulate cancer-specific effector T cells and counteract the activity of tumor-infiltrating immunosuppressive cells. We hypothesised that combining cytokine expression with silencing programmed cell death ligand 1 (PD-L1) could potentiate anticancer immune responses of lentivector vaccines. Thus, we engineered a collection of lentivectors that simultaneously co-expressed an antigen, a PD-L1-silencing shRNA, and various T cell-polarising cytokines, including interferon γ (IFNγ), transforming growth factor β (TGFβ) or interleukins (IL12, IL15, IL23, IL17A, IL6, IL10, IL4). In a syngeneic B16F0 melanoma model and using tyrosinase related protein 1 (TRP1) as a vaccine antigen, we found that simultaneous delivery of IL12 and a PD-L1-silencing shRNA was the only combination that exhibited therapeutically relevant anti-melanoma activities. Mechanistically, we found that delivery of the PD-L1 silencing construct boosted T cell numbers, inhibited in vivo tumor growth and strongly cooperated with IL12 cytokine priming and antitumor activities. Finally, we tested the capacities of our vaccines to counteract tumor-infiltrating myeloid-derived suppressor cell (MDSC) activities ex vivo. Interestingly, the lentivector co-expressing IL12 and the PD-L1 silencing shRNA was the only one that counteracted MDSC suppressive activities, potentially underlying the observed anti-melanoma therapeutic benefit. We conclude that (1) evaluation of vaccines in healthy mice has no significant predictive value for the selection of anticancer treatments; (2) B16 cells expressing xenoantigens as a tumor model are of limited value; and (3) vaccines which inhibit the suppressive effect of MDSC on T cells in our ex vivo assay show promising and relevant antitumor activities.

Assessing T-cell responses in anticancer immunotherapy: Dendritic cells or myeloid-derived suppressor cells?

Since dendritic cells operate as professional antigen-presenting cells (APCs) and hence are capable of jumpstarting the immune system, they have been exploited to develop a variety of immunotherapeutic regimens against cancer. In the few past years, myeloid-derived suppressor cells (MDSCs) have been shown to mediate robust immunosuppressive functions, thereby inhibiting tumor-targeting immune responses. Thus, we propose that the immunomodulatory activity of MDSCs should be carefully considered for the development of efficient anticancer immunotherapies.

PD-L1/PD-1 Co-Stimulation, a Brake for T cell Activation and a T cell Differentiation Signal

For T cell activation, three signals have to be provided from the antigen presenting cell; Signal 1 (antigen recognition), signal 2 (co-stimulation) and signal 3 (cytokine priming). Blocking negative co-stimulation during antigen presentation to T cells is becoming a promising therapeutic strategy to enhance cancer immunotherapy. Here we will focus on interference with PD-1/PD-L1 negative co-stimulation during antigen presentation to T cells as a therapeutic approach. We will discuss the potential mechanisms and the therapeutic consequences by which interference/inhibition with this interaction results in anti-tumour immunity. Particularly, we will comment on whether blocking negative co-stimulation provides differentiation signals to T cells undergoing antigen presentation. A major dogma in immunology states that T cell differentiation signals are given by cytokines and chemokines (signal 3) rather than co-stimulation (signal 2). We will discuss whether this is the case when blocking PD-L1/PD-1 negative co-stimulation.

IFN-α inhibits telomerase in human CD8+ T cells by both hTERT downregulation and induction of p38 MAPK signaling

The cytokine IFN-α is secreted during viral infections and has been shown to inhibit telomerase activity and accelerate T cell differentiation in vivo. However, the mechanism for this inhibition is not clear. In this study, we show that IFN-α inhibits both the transcription and translation of human telomerase reverse transcriptase (hTERT), the catalytic component of telomerase, in activated CD8+ T cells. This was associated with increased activity of the repressor of hTERT transcription E2 transcription factor and decreased activation of NF-κB that promotes hTERT transcription. However IFN-α did not affect the translocation of hTERT from the cytoplasm to the nucleus. IFN-α also inhibits AKT kinase activation but increases p38 MAPK activity, and both of these events have been shown previously to inhibit telomerase activity. Addition of BIRB796, an inhibitor of p38 activity, to IFN-α–treated cells reversed, in part, the inhibition of telomerase by this cytokine. Therefore, IFN-α can inhibit the enzyme telomerase in CD8+ T cells by transcriptional and posttranslational mechanisms. Furthermore, the addition of IFN-α to CD8+CD27+CD28+ T cells accelerates the loss of both these costimulatory molecules. This suggests that persistent viral infections may contribute to the accumulation of highly differentiated/senescent CD8+CD27−CD28− T cells during aging by promoting IFN-α secretion during repeated episodes of viral reactivation.

Immune modulation by genetic modification of dendritic cells with lentiviral vectors

Our work over the past eight years has focused on the use of HIV-1 lentiviral vectors (lentivectors) for the genetic modification of dendritic cells (DCs) to control their functions in immune modulation. DCs are key professional antigen presenting cells which regulate the activity of most effector immune cells, including T, B and NK cells. Their genetic modification provides the means for the development of targeted therapies towards cancer and autoimmune disease. We have been modulating with lentivectors the activity of intracellular signalling pathways and co-stimulation during antigen presentation to T cells, to fine-tune the type and strength of the immune response. In the course of our research, we have found unexpected results such as the surprising immunosuppressive role of anti-viral signalling pathways, and the close link between negative co-stimulation in the immunological synapse and T cell receptor trafficking. Here we review our major findings and put them into context with other published work.