Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disease characterized by platelet defects and oculocutaneous albinism. Individuals with HPS type 2 (HPS2) lack the cytosolic adaptor protein 3 (AP-3) involved in lysosomal sorting, and are also immunodeficient. Here we characterize an HPS2 mutation and demonstrate that AP-3 deficiency leads to a loss of cytotoxic T lymphocyte (CTL)-mediated cytotoxicity. Although the lysosomal protein CD63 was mislocalized to the plasma membrane, perforin and granzymes were correctly localized to the lytic granules in AP-3-deficient CTLs. However, the lytic granules of AP-3-deficient CTLs were enlarged and were unable to move along microtubules and dock within the secretory domain of the immunological synapse. These data show that AP-3 is essential for polarized secretion from CTLs.

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Vaccinia virus (VACV) is the vaccine that was used to eradicate smallpox and is being developed as a recombinant vaccine for other pathogens. Removal of genes encoding immunomodulatory proteins expressed by VACV may enhance virus immunogenicity and improve its potential as a vaccine. Protein A41 is a candidate for removal, having sequence similarity to the VACV chemokine-binding protein, vCKBP, and an association with reduced inflammation during dermal infection. Here, it is shown that, at low doses, VACV strain Western Reserve (WR) lacking A41L (vDA41L) was slightly more virulent than wild-type and revertant controls after intranasal infection of BALB/c mice. The primary immune response to vDA41L was marked by an increase in the percentage of VACV-specific gamma interferon-producing CD8 + T cells and enhancement of cytotoxic T-cell responses in the spleen. However, this augmentation of cellular response was not seen in lung infiltrates. Splenic CD8 + T-cell responses were also enhanced when VACV strain modified vaccinia virus Ankara (MVA) lacking A41L was used to immunize mice. Lastly, immunization with VACV MVA lacking A41L provided better protection than control viruses to subsequent challenge with a 300 LD 50 dose of VACV WR. This study provides insight into the immunomodulatory role of A41 and suggests that MVA lacking A41 may represent a more efficacious vaccine.

The vaccinia virus (VACV) strain Western Reserve C16 protein has been characterized and its effects on virus replication and virulence have been determined. The C16L gene is present in the inverted terminal repeat and so is one of the few VACV genes that are diploid. The C16 protein is highly conserved between different VACV strains, and also in the orthopoxviruses variola virus, ectromelia virus, horsepox virus and cowpox virus. C16 is a 37.5 kDa protein, which is expressed early during infection and localizes to the cell nucleus and cytoplasm of infected and transfected cells. The loss of the C16L gene had no effect on virus growth kinetics but did reduce plaque size slightly. Furthermore, the virulence of a virus lacking C16L (vΔC16) was reduced in a murine intranasal model compared with control viruses and there were reduced virus titres from 4 days post-infection. In the absence of C16, the recruitment of inflammatory cells in the lung and bronchoalveolar lavage was increased early after infection (day 3) and more CD4+ and CD8+ T cells expressed the CD69 activation marker. Conversely, late after infection with vΔC16 (day 10) there were fewer T cells remaining, indicating more rapid clearance of infection. Collectively, these data indicate that C16 diminishes the immune response and is an intracellular immunomodulator.

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.