Granules Of CD8 Research Articles

Endocytosis of cytotoxic granules in CD8 T lymphocytes is required for multiple killing of target cells (IRM14P.448)

Abstract Cytotoxic (CD8+) T lymphocytes (CTLs) eliminate tumorigenic and virus-infected target cells by releasing the contents of cytotoxic granules (CGs) at the immunological synapse. Even though CTLs have only a limited number of pre-existing fusogenic CGs, they can efficiently kill multiple target cells upon activation. We therefore hypothesized that, following CG fusion, the remaining components are recycled and subsequently refilled with cytotoxic substances to become available for the next round of fusion, thus enabling efficient multiple killing. We visualized endogenous CGs and their recycling in real-time in CTLs from synaptobrevin2-mRFP knock-in mice by fluorescently-labeled anti-RFP antibodies. We found that endocytosis (recycling) of CGs was blocked by Dynasore, an inhibitor of dynamin. Additionally, clathrin was colocalized with endocytosing CGs at the plasma membrane following dynasore pretreatment. Thus, endocytosis of CGs is clathrin- and dynamin-dependent. Importantly, although CTLs pre-treated with Dynasore showed no immediate effect on CG exocytosis, they exhibited a 50% deficiency in killing target cells after 2 hours of incubation. Our data strongly suggest that recycling of CGs plays an important role in CTLs for executing multiple killing of target cells.

Abundant expression of granzyme A, but not perforin, in granules of CD8+ T cells in GALT: implications for immune control of HIV-1 infection.

Because GALT is a major portal of entry for HIV-1 and reservoir for viral replication, we hypothesized that an ineffective cellular immune response in intestinal mucosa might partially explain the failure of immune control in AIDS. In this study, we demonstrate that the vast majority of CD8+ T cells in rectal tissue, including HIV-1-specific cells, fail to express the cytolytic protein, perforin. However, rectal CD8+ T cells do express granzyme A, and are also capable of releasing IFN-gamma upon stimulation with cognate peptide. Confocal microscopy showed that granzyme A was located in intracellular granules in the absence of perforin. The majority of rectal CD8+ T cells exhibit an effector memory phenotype, expressing CD45RO but not CCR7. Quantitative real-time PCR analysis demonstrated that perforin RNA is expressed in rectal CD8+ T cells from healthy and HIV-1-positive individuals. In HIV-1-positive individuals, similar amounts of perforin RNA were detected in CD8+ T cells from rectal tissue and PBMC, despite a relative absence of perforin protein in rectal tissue. These findings demonstrate an important difference in perforin expression between CD8+ T cells in blood and mucosa. Furthermore, the relative absence of armed effector cells may serve to protect the integrity of rectal mucosa under normal conditions, but might also provide an early advantage to HIV-1 and other sexually transmitted viruses.

HIV virions and HIV infection in vitro are unaffected by human granzymes A and B.

Granzymes are a family of serine proteinases commonly found in the granules of CD8+ T cells. In HIV infection, CD8+ cells show cytotoxic and noncytotoxic antiviral activities. The latter is mediated, at least in part, by a secreted CD8+ cell antiviral factor, CAF. Because of the antiviral nature of CD8+ cells, we examined the potential anti-HIV activity of free granzymes that can be found in CD8+ cell culture fluids. Pretreatment of CD4+ T cells with granzyme A or granzyme B had no effect on their susceptibility to infection with HIV, nor did incubation of the granzymes with HIV virions alter their infectivity. Continuous culture of acutely infected CD4+ T cells with granzyme A or B showed no effect on cell viability or the replication of HIV. The findings of this study suggest that free granzymes do not control HIV infection and spread in CD4+ T cells.

Perforin is not co-expressed with granzyme A within cytotoxic granules in CD8 T lymphocytes present in lymphoid tissue during chronic HIV infection.

Residual HIV-1-infected cells are poorly eliminated from lymphoid tissue (LT) reservoirs by effector cytotoxic T lymphocytes (eCTL) despite antiretroviral therapy. Perforin and granzyme A (grA) constitute major effector molecules within eCTL granules that induce apoptosis and lysis of virally infected cells. Expression of perforin and grA was studied at the single cell level in LT and blood from 16 patients infected with HIV-1 (stage A1-C) who were not taking antiretroviral therapy. Immunohistochemical analysis by in situ imaging of cells from blood and LT. Quantitative in situ imaging showed that perforin-expressing CD8 T cells comprised 0.3-1.5% of total cells within the LT from recent HIV-1 seroconverters, while grA was found in 2.1-7.2% of total cells. However, despite high-level grA upregulation (1.5-4.5% of total cells) compared with that in non-infected individuals (0.4-0.9%), perforin expression remained low (< 0.1% of total cells) (P < 0.02) in LT from patients with chronic HIV-1 infection (stage A2-C). This contrasted with findings in peripheral blood mononuclear cells (PBMC) from the same HIV-1 infected cohort where perforin was detected in 13-31% of all PBMC, which was 10- to 100-fold higher than in lymphoid tissue (P < 0.001); grA was found in 14-32% of total PBMC. Two-colour staining showed that granular expression of perforin and grA was restricted to CD8 T cells in over 90% of total cells in both LT and blood. These findings indicate that cytotoxic perforin expression is impaired at local sites of HIV replication within lymphoid tissue. Since perforin is required together with grA for granule-mediated cytolysis, the low perforin expression in the LT may limit the ability of eCTL to eliminate HIV-1 infected cells in lymphoid tissue.