Gillian M. Griffiths

Fratricide among CD8+ T Lymphocytes Naturally Infected with Human T Cell Lymphotropic Virus Type I

Infection and gene expression by the human T lymphotropic virus type I (HTLV-I) in vivo have been thought to be confined to CD4(+) T lymphocytes. We show here that, in natural HTLV-I infection, a significant proportion of CD8(+) T lymphocytes are infected by HTLV-I. Interestingly, HTLV-I-specific but not Epstein-Barr virus-specific CD8(+) T lymphocytes were shown to be infected. Furthermore, HTLV-I protein expression in naturally infected CD8(+) T lymphocytes renders them susceptible to fratricide mediated by autologous HTLV-I-specific CD8(+) T lymphocytes. Fratricide among virus-specific CTLs could impair the immune control of HTLV-I and possibly other lymphotropic viruses.

Analysis of the Lysosomal Storage Disease Chediak–Higashi Syndrome

Chediak–Higashi syndrome (CHS) is a rare autosomal recessive disorder of human, mouse (beige) and other mammalian species. The same genetic defect was found to result in the disease in all species identified, permitting a positional cloning approach using the mouse model beige to identify the responsible gene. The CHS gene was cloned and mutations identified in affected species. This review discusses the clinical features of CHS contrasting features seen in similar syndromes. The possible functions of the protein encoded by the CHS/beige gene are discussed, along with the alterations in cellular physiology seen in mutant cells.

The secretory synapse: the secrets of a serial killer

Summary:  Cytotoxic T lymphocytes (CTLs) destroy their targets by a process involving secretion of specialized granules. The interactions between CTLs and target can be very brief; nevertheless, adhesion and signaling proteins segregate into an immunological synapse. Secretion occurs in a specialized secretory domain. Use of live and fixed cell microscopy allows this secretory synapse to be visualized both temporally and spatially. The combined use of confocal and electron microscopy has produced some surprising findings, which suggest that the secretory synapse may be important both in delivering the lethal hit and in facilitating membrane transfer from target to CTL. Studies on the secretory synapse in wild‐type and mutant CTLs have been used to identify proteins involved in secretion. Further clues as to the signals required for secretion are emerging from comparisons of inhibitory and activating synapses formed by natural killer cells.

Novel Munc13-4 mutations in children and young adult patients with haemophagocytic lymphohistiocytosis.

Familial haemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous disorder characterised by constitutive defects in cellular cytotoxicity resulting in fever, hepatosplenomegaly and cytopenia, and the outcome is fatal unless treated by chemoimmunotherapy followed by haematopoietic stem-cell transplantation. Since 1999, mutations in the perforin gene giving rise to this disease have been identified; however, these account only for 40% of cases. Lack of a genetic marker hampers the diagnosis, suitability for transplantation, selection of familial donors, identification of carriers, genetic counselling and prenatal diagnosis. Mutations in the Munc13–4 gene have recently been described in patients with FHL. We sequenced the Munc13–4 gene in all patients with haemophagocytic lymphohistiocytosis not due to PRF1 mutations. In 15 of the 30 families studied, 12 novel and 4 known Munc13–4 mutations were found, spread throughout the gene. Among novel mutations, 2650C→T introduced a stop codon; 441del A, 532del C, 3082del C and 3226ins G caused a frameshift, and seven were mis sense mutations. Median age of diagnosis was 4 months, but six patients developed the disease after 5 years of age and one as a young adult of 18 years. Involvement of central nervous system was present in 9 of 15 patients, activity of natural killer cells was markedly reduced or absent in 13 of 13 tested patients. Chemo-immunotherapy was effective in all patients. Munc13–4 mutations were found in 15 of 30 patients with FHL without PRF1 mutations. Because these patients may develop the disease during adolescence or even later, haematologists should include FHL2 and FHL3 in the differential diagnosis of young adults with fever, cytopenia, splenomegaly and hypercytokinaemia.

Secretory Lysosome Biogenesis in Cytotoxic T Lymphocytes from Normal and Chediak Higashi Syndrome Patients

The lytic proteins mediating target cell killing are stored in the lysosomes of activated cytotoxic T lymphocytes (CTL) and are secreted upon recognition of a target cell. These secretory lysosomes cannot be detected in resting T lymphocytes. Interaction of a resting cell with a target cell activates de novo formation of secretory lysosomes. CTL clones in culture mimic this behaviour, and so provide an ideal system for studying secretory lysosome biogenesis and maturation. In the genetic disease, Chediak Higashi syndrome (CHS), all lysosomes in the cells are enlarged and reduced in number compared with wild‐type (WT) cells. We have used CTL from this disease to study secretory lysosome biogenesis and maturation. We show that at early stages after activation the secretory lysosomes are identical in WT and mutant cells, and that delivery of proteins to the secretory lysosome along the biosynthetic and endocytic pathways is normal in the mutant cells. With time, the lysosomes in the mutant cells aggregate, become larger and fewer in number and eventually form giant structures. Our results show that the initial steps of secretory lysosome formation are normal in CHS, but that the organelles subsequently fuse together during cell maturation to form the giant secretory lysosomes.

Slp1 and Slp2-a Localize to the Plasma Membrane of CTL and Contribute to Secretion from the Immunological Synapse

Rab27a is required for polarized secretion of lysosomes from cytotoxic T lymphocytes (CTLs) at the immunological synapse. A series of Rab27a‐interacting proteins have been identified; however, only Munc13‐4 has been found to be expressed in CTL. In this study, we screened for expression of the synaptotagmin‐like proteins (Slps): Slp1/JFC1, Slp2‐a/exophilin4, Slp3‐a, Slp4/granuphilin, Slp5 and rabphilin in CTL. We found that both Slp1 and Slp2‐a are expressed in CTL. Isoforms of Slp2‐a in CTL showed variation of the linker region but conserved the C2A and C2B and Slp homology (SHD) domains. Both Slp1 and Slp2‐a interact with Rab27a in CTL, and Slp2‐a, but not Slp1, is rapidly degraded when Rab27a is absent. Slp2‐a contains PEST‐like sequences within its linker region, which render it susceptible to degradation. Both Slp1 and Slp2‐a localize predominantly to the plasma membrane of both human and mouse CTLs, and we show that Slp2‐a can focus tightly at the immunological synapse formed with a target cell. Individual knockouts of either Slp2‐a or Slp1 fail to impair CTL‐mediated killing of targets; however, overexpression of a dominant‐negative construct consisting of the SHD of Slp2‐a, which is 56% identical to that of Slp1, reduces target cell death, suggesting that both Slp1 and Slp2‐a contribute to secretory lysosome exocytosis from CTL. These results suggest that both Slp1 and Slp2‐a may form part of a docking complex, capturing secretory lysosomes at the immunological synapse.

Microtubule-Dependent Transport of Secretory Vesicles in RBL-2H3 Cells

Antigen‐mediated activation of mast cells results in Ca2+‐dependent exocytosis of preformed mediators of the inflammatory response. To investigate the role of secretory vesicle motility in this response, we have performed time‐lapse confocal microscopy on RBL‐2H3 cells transfected with a green fluorescent protein‐Fas ligand fusion protein (GFP‐FasL). Green fluorescent protein‐labeled vesicles exhibit rapid, bidirectional movement in both resting and activated cells and can be localized adjacent to microtubules. Colchicine treatment inhibits the motility of secretory vesicles as measured by fluorescence recovery after photobleaching (FRAP). Colchicine also inhibits both the extent and the rate of exocytosis triggered by receptor activation or by Ca2+ ionophore, demonstrating that microtubule‐dependent movement of secretory vesicles plays an important role in the exocytic response.

What's special about secretory lysosomes?

Lysosomes are organelles specialised for their role in intracellular protein degradation. A small number of cell types also use their lysosomes as regulated secretory organelles. These secretory lysosomes package additional secretory products, respond to extracellular stimuli and fuse with the plasma membrane to release their contents. Recent research has identified unique components of the secretory machinery in these cells. However, studies on conventional lysosomes in non-secretory cells reveal that even their lysosomes can fuse with the plasma membrane in response to membrane damage. What then is special about secretory lysosomes?