Understanding cytotoxic T lymphocyte function using models of primary immunodeficiencies

CD8+ cytotoxic T lymphocytes (CTLs) are critical for the elimination of virally-infected cells, and defects in CTL responses can lead to primary immunodeficiencies and secondary lymphoproliferative syndromes. One such defect is caused by mutations in the gene encoding Inducible T cell Kinase (ITK), a kinase that serves as an amplifier of T cell receptor (TCR) signaling. Patients with mutations in ITK develop lymphoproliferative disease associated with susceptibility to viral infections. We found CTLs from ITK-deficient mice exhibit impaired killing of multiple different targets, indicating that ITK-deficiency leads to global defects in cytolysis. Treating WT CTLs with an ITK-specific inhibitor during cytolysis assays could reproduce impaired killing, suggesting that these defects were not necessarily due to altered T cell development or CTL differentiation. To further evaluate this killing defect, we examined the discrete steps involved in CTL activity, including TCR-triggered adherence to cells, immunological synapse formation, centrosome polarization, and degranulation inducing cytolysis in targets. Although early events following TCR-mediated target cell engagement, such as actin ring formation and polarization, were intact in ITK-deficient CTLs, we found defects in degranulation, suggesting ITK may play an unappreciated role in the final stages of killing. Nonetheless, prolonged culture of ITK-deficient CTLs in IL-2 could rescue defects in degranulation, similar to observations in NK cells from certain primary immunodeficiencies in which cytotoxicity is enhanced in culture after IL-2 stimulation. Together these experiments provide clues to novel roles for ITK and TCR signaling in regulating late stages of cytolysis, and further insight into the defects that may account for the susceptibility to viral infections observed in patients with mutations in ITK and TCR signaling components. In parallel work, we also examined the role of actin in regulating degranulation in normal CTLs. While previous work showed a reduction in actin density at the synapse prior to secretion of lytic granules, we found that cortical actin recovers concomitant with the termination of secretion. Disruption of this actin network via treatment with an actin depolymerization agent resulted in a resumed degranulation, suggesting that actin acts as a reversible barrier to prevent lytic granule exocytosis. Furthermore, we provide evidence that degranulation is required to reestablish the actin barrier. Our results suggest that actin is both regulated by, and regulates, degranulation in CTLs. Experiments further revealed a correlation between the recovery of actin and phosphatidylinositol 4,5-biphosphate (PIP2) at the synapse, suggesting that the distribution of phosphatidylinositols in the membrane represent a potential mechanism through which CTLs regulate the density of cortical actin during cytolysis. Our work provides insight into actin-related mechanisms regulating secretion in CTLs, which may preserve serial killing capacity during immune responses

Cortical actin recovery at the immunological synapse leads to termination of lytic granule secretion in cytotoxic T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) eliminate virally infected cells through directed secretion of specialized lytic granules. Because a single CTL can kill multiple targets, degranulation must be tightly regulated. However, how CTLs regulate the termination of granule secretion remains unclear. Previous work demonstrated that centralized actin reduction at the immune synapse precedes degranulation. Using a combination of live confocal, total internal reflection fluorescence, and superresolution microscopy, we now show that, after granule fusion, actin recovers at the synapse and no further secretion is observed. Depolymerization of actin led to resumed granule secretion, suggesting that recovered actin acts as a barrier preventing sustained degranulation. Furthermore, RAB27a-deficient CTLs, which do not secrete cytotoxic granules, failed to recover actin at the synapse, suggesting that RAB27a-mediated granule secretion is required for actin recovery. Finally, we show that both actin clearance and recovery correlated with synaptic phosphatidylinositol 4,5-bisphosphate (PIP2) and that alterations in PIP2 at the immunological synapse regulate cortical actin in CTLs, providing a potential mechanism through which CTLs control cortical actin density. Our work provides insight into actin-related mechanisms regulating CTL secretion that may facilitate serial killing during immune responses

Polyclonal antibody cocktails generated using DNA vaccine technology protect in murine models of orthopoxvirus disease

<p>Abstract</p> <p>Background</p> <p>Previously we demonstrated that DNA vaccination of nonhuman primates (NHP) with a small subset of vaccinia virus (VACV) immunogens (L1, A27, A33, B5) protects against lethal monkeypox virus challenge. The L1 and A27 components of this vaccine target the mature virion (MV) whereas A33 and B5 target the enveloped virion (EV).</p> <p>Results</p> <p>Here, we demonstrated that the antibodies produced in vaccinated NHPs were sufficient to confer protection in a murine model of lethal <it>Orthopoxvirus </it>infection. We further explored the concept of using DNA vaccine technology to produce immunogen-specific polyclonal antibodies that could then be combined into cocktails as potential immunoprophylactic/therapeutics. Specifically, we used DNA vaccines delivered by muscle electroporation to produce polyclonal antibodies against the L1, A27, A33, and B5 in New Zealand white rabbits. The polyclonal antibodies neutralized both MV and EV in cell culture. The ability of antibody cocktails consisting of anti-MV, anti-EV, or a combination of anti-MV/EV to protect BALB/c mice was evaluated as was the efficacy of the anti-MV/EV mixture in a mouse model of progressive vaccinia. In addition to evaluating weight loss and lethality, bioimaging technology was used to characterize the spread of the VACV infections in mice. We found that the anti-EV cocktail, but not the anti-MV cocktail, limited virus spread and lethality.</p> <p>Conclusions</p> <p>A combination of anti-MV/EV antibodies was significantly more protective than anti-EV antibodies alone. These data suggest that DNA vaccine technology could be used to produce a polyclonal antibody cocktail as a possible product to replace vaccinia immune globulin.</p

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Understanding cytotoxic T lymphocyte function using models of primary immunodeficiencies

CD8+ cytotoxic T lymphocytes (CTLs) are critical for the elimination of virally-infected cells, and defects in CTL responses can lead to primary immunodeficiencies and secondary lymphoproliferative syndromes. One such defect is caused by mutations in the gene encoding Inducible T cell Kinase (ITK), a kinase that serves as an amplifier of T cell receptor (TCR) signaling. Patients with mutations in ITK develop lymphoproliferative disease associated with susceptibility to viral infections. We found CTLs from ITK-deficient mice exhibit impaired killing of multiple different targets, indicating that ITK-deficiency leads to global defects in cytolysis. Treating WT CTLs with an ITK-specific inhibitor during cytolysis assays could reproduce impaired killing, suggesting that these defects were not necessarily due to altered T cell development or CTL differentiation. To further evaluate this killing defect, we examined the discrete steps involved in CTL activity, including TCR-triggered adherence to cells, immunological synapse formation, centrosome polarization, and degranulation inducing cytolysis in targets. Although early events following TCR-mediated target cell engagement, such as actin ring formation and polarization, were intact in ITK-deficient CTLs, we found defects in degranulation, suggesting ITK may play an unappreciated role in the final stages of killing. Nonetheless, prolonged culture of ITK-deficient CTLs in IL-2 could rescue defects in degranulation, similar to observations in NK cells from certain primary immunodeficiencies in which cytotoxicity is enhanced in culture after IL-2 stimulation. Together these experiments provide clues to novel roles for ITK and TCR signaling in regulating late stages of cytolysis, and further insight into the defects that may account for the susceptibility to viral infections observed in patients with mutations in ITK and TCR signaling components. In parallel work, we also examined the role of actin in regulating degranulation in normal CTLs. While previous work showed a reduction in actin density at the synapse prior to secretion of lytic granules, we found that cortical actin recovers concomitant with the termination of secretion. Disruption of this actin network via treatment with an actin depolymerization agent resulted in a resumed degranulation, suggesting that actin acts as a reversible barrier to prevent lytic granule exocytosis. Furthermore, we provide evidence that degranulation is required to reestablish the actin barrier. Our results suggest that actin is both regulated by, and regulates, degranulation in CTLs. Experiments further revealed a correlation between the recovery of actin and phosphatidylinositol 4,5-biphosphate (PIP2) at the synapse, suggesting that the distribution of phosphatidylinositols in the membrane represent a potential mechanism through which CTLs regulate the density of cortical actin during cytolysis. Our work provides insight into actin-related mechanisms regulating secretion in CTLs, which may preserve serial killing capacity during immune responses

Inducible T Cell Kinase Regulates the Acquisition of Cytolytic Capacity and Degranulation in CD8+ CTLs.

Patients with mutations in inducible T cell kinase (ITK) are susceptible to viral infections, particularly EBV, suggesting that these patients have defective function of CD8+ CTLs. In this study, we evaluated the effects of ITK deficiency on cytolysis in murine CTLs deficient in ITK, and both human and murine cells treated with an ITK inhibitor. We find that ITK deficiency leads to a global defect in the cytolysis of multiple targets. The absence of ITK both affected CTL expansion and delayed the expression of cytolytic effectors during activation. Furthermore, absence of ITK led to a previously unappreciated intrinsic defect in degranulation. Nonetheless, these defects could be overcome by early or prolonged exposure to IL-2, or by addition of IL-12 to cultures, revealing that cytokine signaling could restore the acquisition of effector function in ITK-deficient CD8+ T cells. Our results provide new insight into the effect of ITK and suboptimal TCR signaling on CD8+ T cell function, and how these may contribute to phenotypes associated with ITK deficiency