Protective immunity afforded by attenuated, PhoP-deficient Mycobacterium tuberculosis is associated with sustained generation of CD4+ T-cell memory.

Definition of protective immunity induced by effective vaccines is important for the design of new pathogen control strategies. Inactivation of the PhoP response-regulator in Mycobacterium tuberculosis results in a highly attenuated strain that demonstrates impressive protective efficacy in pre-clinical models of tuberculosis. In this report we demonstrate that the protection afforded by the M. tuberculosis phoP mutant strain is associated with the long-term maintenance of CD4(+) T-cell memory. Immunization of mice with SO2 resulted in enhanced expansion of M. tuberculosis-specific CD4(+) T cells compared with vaccination with the BCG vaccine, with an increased frequency of these cells persisting at extended time-points after vaccination. Strikingly, vaccination with SO2 resulted in sustained generation of CD4(+) T cells displaying a central memory phenotype, a property not shared by BCG. Further, SO2 vaccination markedly improved the generation of polyfunctional cytokine-secreting CD4(+) T cells compared with BCG vaccination. The improved generation of functionally competent memory T cells by SO2 correlated with augmented recall responses in SO2-vaccinated animals after challenge with virulent M. tuberculosis. This study defines a mechanism for the protective effect of the SO2 vaccine and suggests that deletion of defined virulence networks may provide vaccine strains with potent immuno-stimulatory properties.

Oxidative phosphorylation induces de novo expression of the MHC class I in tumor cells through the ERK5 pathway.

Most cancer cells use anaerobic-like glycolysis to generate energy instead of oxidative phosphorylation. They also avoid recognition by CTLs, which occurs primarily through decreasing the level of MHC class I (MHC-I) at the cell surface. We find that the two phenomena are linked; culture conditions that force respiration in leukemia cells upregulate MHC-I transcription and protein levels at the cell surface, whereas these decrease in cells forced to perform fermentation as well as in leukemia cells lacking a functional mitochondrial respiratory chain. Forced respiration leads to increased expression of the MAPK ERK5, which activates MHC-I gene promoters, and ERK5 accumulation in mitochondria. Respiration-induced MHC-I upregulation is reversed upon short hairpin RNA-mediated ERK5 downregulation and by inactive mutants of ERK5. Moreover, short hairpin RNA for ERK5 leukemia cells do not tolerate forced respiration. Thus, the expression of ERK5 and MHC-I is linked to cell metabolism and notably diminished by the metabolic adaptations found in tumor cells.

Protein kinase C-theta is required for NK cell activation and in vivo control of tumor progression.

Protein kinase C-theta (PKCtheta) was initially isolated as an important PKC isoform expressed in T cells, although its expression is not restricted to these cells. Despite the central function of PKCtheta in several immune responses, its role in the antitumor response against MHC class I (MHC-I)-negative cells has not been investigated. This is an important issue because most tumor cells growing in vivo down-regulate MHC-I expression to escape the CTL-mediated response. In the present work, we show that in vivo development of a MHC-I-deficient tumor (RMA-S) is much favored in PKCtheta(-/-) mice compared with wild-type mice. This is associated with a reduced recruitment of NK cells to the site of tumor development and a reduced activation status of recruited NK cells. This correlates with a reduced ex vivo and in vivo cytotoxic potential of NK cells isolated from PKCtheta(-/-) mice treated with polyinosinic:polycytidylic acid. Consistently, polinosinic:cytidilic acid treatment induces PKCtheta expression and activation of its enzymatic activity in NK cells in an indirect manner. These observations underline the relevance of PKCtheta as a key molecule in NK cell-mediated antitumor immune surveillance.

Granzyme B of cytotoxic T cells induces extramitochondrial reactive oxygen species production via caspase-dependent NADPH oxidase activation.

Induction of reactive oxygen species (ROS) is a hallmark of granzyme B (gzmB)-mediated pro-apoptotic processes and target cell death. However, it is unclear to what extent the generated ROS derive from mitochondrial and/or extra-mitochondrial sources. To clarify this point, we have produced a mutant EL4 cell line, termed EL4-rho(0), which lacks mitochondrial DNA, associated with a decreased mitochondrial membrane potential and a defective ROS production through the electron transport chain of oxidative phosphorylation. When incubated with either recombinant gzmB plus streptolysin or ex vivo gzmB(+) cytotoxic T cells, EL4-rho(0) cells showed phosphatydylserine translocation, caspase 3 activation, Bak conformational change, cytochrome c release and apoptotic morphology comparable to EL4 cells. Moreover, EL4-rho(0) cells produced ROS at levels similar to EL4 under these conditions. GzmB-mediated ROS production was almost totally abolished in both cell lines by the pan-caspase inhibitor, Z-VAD-fmk. However, addition of apocynin, a specific inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, led to a significant reduction of ROS production and cell death only in EL4-rho(0) but not EL4 cells. These data suggest that gzmB-induced cell death is accompanied by a caspase-dependent pathway of extra-mitochondrial ROS production, most probably through activation of NADPH oxidase.

Cell cycle regulation by FasL and Apo2L/TRAIL in human T-cell blasts. Implications for autoimmune lymphoproliferative syndromes.

The Fas-FasL pathway plays an important role in the homeostasis of mature lymphocytes, with defects causing autoimmune lymphoproliferative syndromes (ALPS). Human T-cell blasts are not sensitive to FasL or Apo2L/TRAIL-induced apoptosis unless they get reactivated, but either of those ligands inhibits their growth in the absence of cell death induction due to a cell cycle arrest in S-G2/M. In the present work, we have studied the mechanism(s) by which FasL or Apo2L/TRAIL regulate T-cell blast cell cycle in healthy donors and in two types of ALPS patients. Our data indicate that in human CD8+ T-cell blasts, Fas ligation, and especially Apo2L/TRAIL induce the p53-dependent decrease in cyclin-B1 levels. However, the induction of the negative cell cycle regulator p21WAF1 by FasL or Apo2L/TRAIL in either CD4+ or CD8+ T-cell blasts seems to be the main regulatory mechanism. This mechanism is dependent on caspase activation and on H2O2 generation. The increase in p21 levels by FasL or Apo2L/TRAIL is concomitant with p53 increases only in CD8+ T-cell blasts, with p21 levels maintained high for longer times than p53 levels. In CD4+ T-cell blasts p21 levels are controlled through a transient and p53-independent mechanism. The present results suggest that the etiology of ALP syndromes could be related not only to defects in apoptosis induction, but also in cell cycle regulation.

Autoimmune lymphoproliferative syndrome (ALPS) in a patient with a new germline Fas gene mutation.

Autoimmune lymphoproliferative syndrome (ALPS) is a rare genetic disorder characterized by chronic lymphoproliferation, autoimmune manifestations and expansion of TCRalphabeta+CD4-CD8- lymphocytes. The main pathogenic factor is a defective Fas-mediated apoptosis generally caused by mutations in the Fas gene. This report describes a new heterozygous Fas gene mutation in a boy with clinical and immunological features of ALPS. In vitro, T-cell blasts from the patient are completely resistant to the effects on the anti-Fas cytotoxic mAb CH-11, they also have a higher proliferation rate than T cells from healthy donors, while PHA-induced AICD is normal. The location of the mutation (I246S) found in the intracytoplasmic death domain, and the conservation of that residue in four different species from human suggest that I246 is an essential amino acid for Fas function. The patient has inherited the mutation from his father who also shows defective Fas-mediated apoptosis but the clinical and immunological manifestations are much less severe. These results provide evidence that the penetrance of genetic defects in Fas is variable and that other factors may influence the phenotype of the disease.

Construction, characterization and preclinical evaluation of MTBVAC, the first live-attenuated M. tuberculosis-based vaccine to enter clinical trials.

The development of a new tuberculosis vaccine is an urgent need due to the failure of the current vaccine, BCG, to protect against the respiratory form of the disease. MTBVAC is an attenuated Mycobacterium tuberculosis vaccine candidate genetically engineered to fulfil the Geneva consensus requirements to enter human clinical trials. We selected a M. tuberculosis clinical isolate to generate two independent deletions without antibiotic-resistance markers in the genes phoP, coding for a transcription factor key for the regulation of M. tuberculosis virulence, and fadD26, essential for the synthesis of the complex lipids phthiocerol dimycocerosates (DIM), one of the major mycobacterial virulence factors. The resultant strain MTBVAC exhibits safety and biodistribution profiles similar to BCG and confers superior protection in preclinical studies. These features have enabled MTBVAC to be the first live attenuated M. tuberculosis vaccine to enter clinical evaluation.

Protein Kinase C-θ (PKC-θ) in Natural Killer Cell Function and Anti-Tumor Immunity.

The protein kinase C-θ (PKCθ), which is essential for T cell function and survival, is also required for efficient anti-tumor immune surveillance. Natural killer (NK) cells, which express PKCθ, play a prominent role in this process, mainly by elimination of tumor cells with reduced or absent major histocompatibility complex class-I (MHC-I) expression. This justifies the increased interest of the use of activated NK cells in anti-tumor immunotherapy in the clinic. The in vivo development of MHC-I-deficient tumors is much favored in PKCθ(-/-) mice compared with wild-type mice. Recent data offer some clues on the mechanism that could explain the important role of PKCθ in NK cell-mediated anti-tumor immune surveillance: some studies show that PKCθ is implicated in signal transduction and anti-tumoral activity of NK cells elicited by interleukin (IL)-12 or IL-15, while others show that it is implicated in NK cell functional activation mediated by certain killer-activating receptors. Alternatively, the possibility that PKCθ is involved in NK cell degranulation is discussed, since recent data indicate that it is implicated in microtubule-organizing center polarization to the immune synapse in CD4(+) T cells. The implication of PKC isoforms in degranulation has been more extensively studied in cytotoxic T lymphocyte, and these studies will be also summarized.

Attenuated Mycobacterium tuberculosis SO2 vaccine candidate is unable to induce cell death.

It has been proposed that Mycobacterium tuberculosis virulent strains inhibit apoptosis and trigger cell death by necrosis of host macrophages to evade innate immunity, while non-virulent strains induce typical apoptosis activating a protective host response. As part of the characterization of a novel tuberculosis vaccine candidate, the M. tuberculosis phoP mutant SO2, we sought to evaluate its potential to induce host cell death. The parental M. tuberculosis MT103 strain and the current vaccine against tuberculosis Bacillus Calmette-Guérin (BCG) were used as comparators in mouse models in vitro and in vivo. Our data reveal that attenuated SO2 was unable to induce apoptotic events neither in mouse macrophages in vitro nor during lung infection in vivo. In contrast, virulent MT103 triggers typical apoptotic events with phosphatidylserine exposure, caspase-3 activation and nuclear condensation and fragmentation. BCG strain behaved like SO2 and did not induce apoptosis. A clonogenic survival assay confirmed that viability of BCG- or SO2-infected macrophages was unaffected. Our results discard apoptosis as the protective mechanism induced by SO2 vaccine and provide evidence for positive correlation between classical apoptosis induction and virulent strains, suggesting apoptosis as a possible virulence determinant during M. tuberculosis infection.