Granule-dependent Mechanism Research Articles

BTK Inhibitors Impair Platelet-Mediated Antifungal Activity.

In recent years, the introduction of new drugs targeting Bruton’s tyrosine kinase (BTK) has allowed dramatic improvement in the prognosis of patients with chronic lymphocytic leukemia (CLL) and other B-cell neoplasms. Although these small molecules were initially considered less immunosuppressive than chemoimmunotherapy, an increasing number of reports have described the occurrence of unexpected opportunistic fungal infections, in particular invasive aspergillosis (IA). BTK represents a crucial molecule in several signaling pathways depending on different immune receptors. Based on a variety of specific off-target effects on innate immunity, namely on neutrophils, monocytes, pulmonary macrophages, and nurse-like cells, ibrutinib has been proposed as a new host factor for the definition of probable invasive pulmonary mold disease. The role of platelets in the control of fungal growth, through granule-dependent mechanisms, was described in vitro almost two decades ago and is, so far, neglected by experts in the field of clinical management of IA. In the present study, we confirm the antifungal role of platelets, and we show, for the first time, that the exposure to BTK inhibitors impairs several immune functions of platelets in response to Aspergillus fumigatus, i.e., the ability to adhere to conidia, activation (as indicated by reduced expression of P-selectin), and direct killing activity. In conclusion, our experimental data suggest that antiplatelet effects of BTK inhibitors may contribute to an increased risk for IA in CLL patients.

Potential Antifungal Effects of Human Platelets against Zygomycetes In Vitro

Zygomycosis is increasingly recognized in immunocompromised hosts. We investigated whether platelets become activated after contact with Zygomycetes and adhere to conidial and hyphal structures using immunofluorescence. The platelets' influence on fungal viability was evaluated by assessing hyphal elongation and hyphal damage. Platelets became activated and strongly adhered to conidia and hyphae of Zygomycetes. Platelets induced time dependent damage to hyphae and significantly reduced (P<.05) hyphal elongation. We found that platelets possess antifungal capacities against Zygomycetes based on granule dependent mechanisms and significantly reduce fungal growth and spread, both of which are of major importance in evolving invasive disease.

Antiretroviral compounds affect the granule-dependent mechanisms of lysis in CD8 T cells

Background Cytotoxic T-lymphocytes (CTLs) are essential for suppression of viral replication and, in particular, they have a pivotal role in control of progression of HIV infection. It has been demonstrated that HIV-specific CTL responses are defective in HIV-infected patients undergoing highly active antiretroviral therapy (HAART). In this study we investigated the effects of antiretroviral compounds on the granule-dependent mechanisms of lysis in peripheral blood mononuclear cells (PBMCs).

Effector Functions of Heparin‐Binding Hemagglutinin–Specific CD8+T Lymphocytes in Latent Human Tuberculosis

Most individuals infected with Mycobacterium tuberculosis do not develop tuberculosis (TB) and can be regarded as being protected by an appropriate immune response to the infection. The characterization of the immune responses of individuals with latent TB may thus be helpful in the definition of correlates of protection and the development of new vaccine strategies. The highly protective antigen heparin-binding hemagglutinin (HBHA) induces strong interferon (IFN)- gamma responses during latent, but not active, TB. Because of the recently recognized importance of CD8(+) T lymphocytes in anti-TB immunity, we characterized the CD8(+) T lymphocyte responses to HBHA in subjects with latent TB. HBHA-specific CD8(+) T lymphocytes expressed memory cell markers and synthesized HBHA-specific IFN- gamma . They also restricted mycobacterial growth and expressed cytotoxicity by a granule-dependent mechanism. This activity was associated with the intracellular expression of HBHA-induced perforin. Surprisingly, the perforin-producing CD8(+) T lymphocytes were distinct from the IFN- gamma -producing CD8(+) T lymphocytes. During latent TB, the HBHA-specific CD8(+) T lymphocyte population expresses all 3 effector functions associated with CD8(+) T lymphocyte-mediated protective immune mechanisms, which supports the notion that HBHA may be protective in humans and suggests that markers of HBHA-specific CD8(+) T lymphocyte responses may be useful in the monitoring of protection.

Fas and TNFR1, but not cytolytic granule-dependent mechanisms, mediate clearance of murine liver adenoviral infection

After intravenous injection of replication-deficient adenovirus, hepatocytes are transduced and express high levels of adenovirus-encoded genes. However, adenovirally encoded gene expression is ablated rapidly by CD8+ T-cell-dependent mechanisms. Thus, this model is suitable for examining intrahepatic cytotoxic T lymphocyte (CTL) effector mechanisms. In the present studies, recombinant adenoviruses encoding secreted (human apolipoprotein A-I) or intracellular (beta-galactosidase) gene products were infused into mice with genetic deficiencies affecting the granule exocytosis-, Fas-, or tumor necrosis factor receptor 1 (TNFR1)-mediated pathways of CTL and natural killer cell effector function; the rates of clearance of adenovirus-encoded gene products were assessed. Clearance of secreted or intracellular adenoviral gene products was not delayed in perforin-deficient mice or dipeptidyl peptidase I-deficient mice, which fail to process and activate granzyme A or granzyme B. TNFR1-deficient mice also exhibited no delay in clearance of adenoviral gene products. However, adenoviral clearance from Fas-deficient mice was delayed, and such delays were much greater in mice deficient in both TNFR1 and Fas. In contrast, chimeric mice lacking both hepatic Fas and lymphocyte perforin function exhibited no greater delay in adenoviral clearance than chimeras deficient only in hepatic Fas expression. In conclusion, Fas-dependent mechanisms are required for efficient clearance of virally infected hepatocytes and, in Fas-deficient animals, TNFR1-dependent mechanisms provide an alternative mechanism for hepatic adenovirus clearance. In contrast, perforin- and granule protease-dependent cytotoxicity mechanisms play no apparent role in clearance of adenovirus from the liver.

Identification of a Human HLA-E-Restricted CD8+ T Cell Subset in Volunteers Immunized withSalmonella entericaSerovar Typhi Strain Ty21a Typhoid Vaccine

Our previous studies in volunteers immunized with Salmonella enterica serovar Typhi (S. Typhi) have suggested an important role for CD8+ T cells in host defense. In this study we describe a novel subset of nonclassical human HLA-E-restricted S. Typhi-specific CD8+ T cells derived from PBMC of Ty21a typhoid vaccinees. CD3+CD8+CD4-CD56- T cells effectively killed S. Typhi-infected targets regardless of whether they share classical HLA class I molecules with them, by a FAS-independent, granule-dependent mechanism, as evidenced by induction of granzyme B release and the blocking effects of concanamycin and strontium ions. The expression of HLA-E Ags, but not CD1-a, -b, or -c, on the membrane of S. Typhi-infected targets rendered them susceptible to lysis. Moreover, anti-HLA-E Abs partially blocked these responses. We also demonstrated that presentation of S. Typhi Ags via HLA-E could stimulate IFN-gamma production. Increases in the net frequency of IFN-gamma spot-forming cells were observed in the presence of targets coated with peptides that contain S. Typhi GroEL HLA-E binding motifs. These results demonstrate that HLA-E binds nonamer peptides derived from bacterial proteins and trigger CD8+-mediated lysis and IFN-gamma production when exposed to infected targets, raising the possibility that this novel effector mechanism might contribute to host defense against intracellular bacterial infections.

Granule-dependent mechanisms of lysis are defective in CD8 T cells of HIV-infected, antiretroviral therapy-treated individuals.

HIV-specific cytotoxic T-cell (CTL) responses are defective in HIV-infected patients undergoing antiretroviral therapy (ART). This defect has been attributed to the decreased antigenic burden secondary to ART-associated suppression of HIV-replication, and is responsible for the rebounds of viraemia that occur when patients interrupt therapy. CTL are stimulated by type 1 cytokines and can kill targets via granule-dependent (perforin and granzymes) and -independent (tumour necrosis factor-alpha, CD95) mechanisms. Granule-dependent and granule-independent mechanisms of CTL killing, as well as type 1 cytokine production by CD4 T cells, were analysed in 57 chronically HIV-infected ART-treated or ART-untreated individuals. The results can be summarized as follows: the frequency of gp160 (env)-specific interferon-gamma-secreting CD8 T lymphocytes correlates positively with HIV viraemia in ART-treated and -untreated patients; Env-specific perforin- and granzymes-expressing CD8 T lymphocytes, and Env-stimulated perforin and granzymes mRNA, are reduced in ART-treated patients independently of HIV viral load and of type 1 cytokine production; tumour necrosis factor-alpha production is increased in ART-treated individuals; and Env-specific immature CD8+28+27+ cells are only marginally augmented in ART-treated patients, Similar results are observed in cytomegalovirus-specific CD8 T cells and peripheral blood mononuclear cells. A defect of CTL function that selectively affects the granule-dependent mechanisms of lysis is observed in ART-treated individuals. Because interferon-gamma production is higher in these patients, this could be a defect primarily involving CTL. These data suggest an independence of CD8 T-cell numbers and their lytic ability in HIV-infected, ART-receiving patients. Immunomodulants are needed to successfully treat HIV infection.

Granulysin-dependent killing of intracellular and extracellular Mycobacterium tuberculosis by Vgamma9/Vdelta2 T lymphocytes.

Contribution of Vgamma9/Vdelta2 T lymphocytes to immune protection against Mycobacterium tuberculosis is still a matter of debate. It was reported earlier that Vgamma9/Vdelta2 T lymphocytes kill macrophages harboring live M. tuberculosis through a granule-dependent mechanism that results in killing of intracellular bacilli. This study found that Vgamma9/Vdelta2 T lymphocytes reduce the viability of both extracellular and intracellular M. tuberculosis. Granulysin and perforin, both detected in Vgamma9/Vdelta2 T lymphocytes, play a major role, which indicates that Vgamma9/Vdelta2 T lymphocytes directly contribute to a protective host response against M. tuberculosis infection.

Acute myeloblastic leukemic cells acquire cellular cytotoxicity under genotoxic stress: implication of granzyme B and perforin

Granzyme B (GrB) and perforin (PFN) are the major components of cytoplasmic granules contained in immune cellular effectors. The granule secretory pathway is one of the mechanisms by which these cells exert their cellular cytotoxicity. Recently, it has been reported that GrB and PFN are also present in circulating hemopoietic CD34(+) progenitor cells mobilized by chemotherapy and granulocyte-colony stimulating factor, whereas these proteins are undetected in steady-state peripheral CD34(+) cells. In this study, we hypothesized that anticancer agents may increase GrB and PFN expression in immature myeloid leukemic cells and that these treated leukemic cells become cellular effectors through a granule-dependent mechanism. Our results show that KG1a, HEL, and TF-1 CD34(+) acute myeloblastic leukemia cells expressed both GrB and PFN. Moreover, ionizing radiation, aracytine, and etoposide not only increase GrB and PFN expression but also conferred potent cellular cytotoxicity to these cells toward various cellular targets. Cellular cytotoxicity required cell-cell contact, was not influenced by anti-tumor necrosis factor alpha or anti-Fas blocking antibodies, and was abrogated by GrB inhibitors or antisense. These results suggest that, when exposed to genotoxic agents, immature leukemic cells acquire potent GrB- and PFN-dependent cellular cytotoxicity that can be potentially directed against normal residual myeloid progenitors or immune effectors. (Blood. 2000;96:1914-1920)

Acute myeloblastic leukemic cells acquire cellular cytotoxicity under genotoxic stress: implication of granzyme B and perforin

AbstractGranzyme B (GrB) and perforin (PFN) are the major components of cytoplasmic granules contained in immune cellular effectors. The granule secretory pathway is one of the mechanisms by which these cells exert their cellular cytotoxicity. Recently, it has been reported that GrB and PFN are also present in circulating hemopoietic CD34+ progenitor cells mobilized by chemotherapy and granulocyte-colony stimulating factor, whereas these proteins are undetected in steady-state peripheral CD34+ cells. In this study, we hypothesized that anticancer agents may increase GrB and PFN expression in immature myeloid leukemic cells and that these treated leukemic cells become cellular effectors through a granule-dependent mechanism. Our results show that KG1a, HEL, and TF-1 CD34+acute myeloblastic leukemia cells expressed both GrB and PFN. Moreover, ionizing radiation, aracytine, and etoposide not only increase GrB and PFN expression but also conferred potent cellular cytotoxicity to these cells toward various cellular targets. Cellular cytotoxicity required cell-cell contact, was not influenced by anti-tumor necrosis factor α or anti-Fas blocking antibodies, and was abrogated by GrB inhibitors or antisense. These results suggest that, when exposed to genotoxic agents, immature leukemic cells acquire potent GrB- and PFN-dependent cellular cytotoxicity that can be potentially directed against normal residual myeloid progenitors or immune effectors.

Vgamma9/Vdelta2 T lymphocytes reduce the viability of intracellular Mycobacterium tuberculosis.

An effective immune response against the intracellular pathogen Mycobacterium tuberculosis is strictly dependent on T cell activation. Although this protective response mainly depends on local release of pro-inflammatory cytokines by Th1 CD4(+) T cells, contribution of Vgamma9 / Vdelta2 T lymphocytes to immune protection against this pathogen is suggested by the antimycobacterial reactivity of this subset and its ability to produce large amounts of Th1 cytokines. Here we show that Vgamma9 / Vdelta2 T lymphocytes kill macrophages harboring live M. tuberculosis. The cytotoxic activity of Vgamma9 / Vdelta2 T lymphocytes was not MHC class I or class II restricted but was blocked by anti-TCR monoclonal antibodies, thus indicating that it involved specific interaction between the TCR and the target cell. The cytotoxicity of Vgamma9 / Vdelta2 T lymphocytes was not mediated by TNF-alpha or Fas-Fas ligand, but was shown to occur through a granule-dependent mechanism that resulted in reduction of the viability of intracellular bacilli. Perforin was shown to play an important role in killing of both infected macrophages and intracellular mycobacteria. These data strongly suggest that Vgamma9 / Vdelta2 T lymphocytes contribute to the host defense against M. tuberculosis infection.

An Antimicrobial Activity of Cytolytic T Cells Mediated by Granulysin

Cytolytic T lymphocytes (CTLs) kill intracellular pathogens by a granule-dependent mechanism. Granulysin, a protein found in granules of CTLs, reduced the viability of a broad spectrum of pathogenic bacteria, fungi, and parasites in vitro. Granulysin directly killed extracellular Mycobacterium tuberculosis, altering the membrane integrity of the bacillus, and, in combination with perforin, decreased the viability of intracellular M. tuberculosis. The ability of CTLs to kill intracellular M. tuberculosis was dependent on the presence of granulysin in cytotoxic granules, defining a mechanism by which T cells directly contribute to immunity against intracellular pathogens.