Sarah Booth

The Immunological Synapse of CTL Contains a Secretory Domain and Membrane Bridges

Cytotoxic T lymphocytes (CTL) rapidly destroy their targets. Here we show that although target cell death occurs within 5 min of CTL-target cell contact, an immunological synapse similar to that seen in CD4 cells rapidly forms in CTL, with a ring of adhesion proteins surrounding an inner signaling molecule domain. Lytic granule secretion occurs in a separate domain within the adhesion ring, maintaining signaling protein organization during exocytosis. Live and fixed cell studies show target cell plasma membrane markers are transferred to the CTL as the cells separate. Electron microscopy reveals continuities forming membrane bridges between the CTL and target cell membranes, suggesting a possible mechanism for this transfer.

Invariant NKT cells reduce the immunosuppressive activity of influenza A virus–induced myeloid-derived suppressor cells in mice and humans

Infection with influenza A virus (IAV) presents a substantial threat to public health worldwide, with young, elderly, and immunodeficient individuals being particularly susceptible. Inflammatory responses play an important role in the fatal outcome of IAV infection, but the mechanism remains unclear. We demonstrate here that the absence of invariant NKT (iNKT) cells in mice during IAV infection resulted in the expansion of myeloid-derived suppressor cells (MDSCs), which suppressed IAV-specific immune responses through the expression of both arginase and NOS, resulting in high IAV titer and increased mortality. Adoptive transfer of iNKT cells abolished the suppressive activity of MDSCs, restored IAV-specific immune responses, reduced IAV titer, and increased survival rate. The crosstalk between iNKT and MDSCs was CD1d- and CD40-dependent. Furthermore, IAV infection and exposure to TLR agonists relieved the suppressive activity of MDSCs. Finally, we extended these results to humans by demonstrating the presence of myeloid cells with suppressive activity in the PBLs of individuals infected with IAV and showed that their suppressive activity is substantially reduced by iNKT cell activation. These findings identify what we believe to be a novel immunomodulatory role of iNKT cells, which we suggest could be harnessed to abolish the immunosuppressive activity of MDSCs during IAV infection.

Adaptor protein 3–dependent microtubule-mediated movement of lytic granules to the immunological synapse

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.

Invariant NKT cells modulate the suppressive activity of IL-10-secreting neutrophils differentiated with serum amyloid A

Neutrophils are the main effector cells during inflammation, but they can also control excessive inflammatory responses by secreting anti-inflammatory cytokines. However, the mechanisms that modulate their plasticity remain unclear. We now show that systemic serum amyloid A 1 (SAA-1) controls the plasticity of neutrophil differentiation. SAA-1 not only induced anti-inflammatory interleukin 10 (IL-10)-secreting neutrophils but also promoted the interaction of invariant natural killer T cells (iNKT cells) with those neutrophils, a process that limited their suppressive activity by diminishing the production of IL-10 and enhancing the production of IL-12. Because SAA-1-producing melanomas promoted differentiation of IL-10-secreting neutrophils, harnessing iNKT cells could be useful therapeutically by decreasing the frequency of immunosuppressive neutrophils and restoring tumor-specific immune responses.

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.

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.

Prostaglandin E2 suppresses the differentiation of retinoic acid–producing dendritic cells in mice and humans

Prostaglandin E2 inhibits the expression of retinal dehydrogenase, thus inhibiting retinoic acid production and the priming of gut-tropic T cells by dendritic cells.

A Role for Rab7 in the Movement of Secretory Granules in Cytotoxic T Lymphocytes

Cytotoxic T lymphocytes (CTL) are potent killers of virally infected and tumorigenic cells. Upon recognition of target cells, CTL undergo polarized secretion of secretory lysosomes at the immunological synapse (IS) that forms between CTL and target. However, the molecular machinery involved in the polarization of secretory lysosomes is still largely uncharacterized. In this paper, we investigated the role of Rab7 in the polarization of secretory lysosomes. We show that silencing of Rab7 by RNA interference reduces the ability of CTL to kill targets. GTP‐bound Rab7 and Rab interacting lysosomal protein, RILP, interact and both localize to secretory lysosomes in CTL. Over‐expression of RILP recruits dynein to the membranes of secretory lysosomes and triggers their movement toward the centrosome. Together, these results suggest that Rab7 may play a role in secretory lysosome movement toward the centrosome by interacting with RILP to recruit the minus‐end motor, dynein.

Two patients with Hermansky Pudlak syndrome type 2 and novel mutations in AP3B1

Hermansky Pudlak syndrome type 2 (HPS2) is a rare disorder associated with mutations in the Adaptor Protein 3 (AP-3) complex, which is involved in sorting transmembrane proteins to lysosomes and related organelles. We now report 2 unrelated subjects with HPS2 who show a characteristic clinical phenotype of oculocutaneous albinism, platelet and T-lymphocyte dysfunction and neutropenia. The subjects were homozygous for different deletions within AP3B1 (g.del180242-180866, c.del153-156), which encodes the AP-3β3A subunit, resulting in frame shifts and introduction of nonsense substitutions (p.E693fsX13, p.E52fsX11). In the subject with p.E693fsX13, this resulted in expression of a truncated variant β3A protein. Cytotoxic T-lymphocyte (CTL) clones from both study subjects showed increased cell-surface expression of CD63 and reduced cytotoxicity. Platelets showed impaired aggregation and reduced uptake of 3H-serotonin. These findings are consistent with CTL granule and platelet dense granule defects, respectively. This report extends the clinical and laboratory description of HPS2.

Normal Lytic Granule Secretion by Cytotoxic T Lymphocytes Deficient in BLOC‐1, ‐2 and ‐3 and Myosins Va, VIIa and XV

Melanocytes and cells of the immune system share an unusual secretory mechanism which uses the lysosome as a regulated secretory organelle. Recently, a number of the proteins required for these ‘secretory lysosomes’ to undergo exocytosis have been identified. These include Rab27a, Lyst, Rab geranyl geranyl transferase and the adapter protein complex AP‐3. Patients lacking any of these proteins are characterized by the rare combination of albinism and immunodeficiency, revealing roles for these proteins in both melanocyte and immune cell secretion. In order to ask how far the link between albinism and immunodeficiency extends we have examined cytotoxic T‐lymphocyte (CTL) secretion from two BLOC‐3‐deficient patients and seven different mouse models of Hermansky–Pudlak syndrome, all of which display defects in pigmentation and platelet function. We find that CTL function is normal in HPS patients and pale‐ear mice deficient in BLOC‐3, pallid, muted and sandy mice deficient in BLOC‐1, ruby‐eye mice deficient in BLOC‐2 and buff mice deficient in Vps33a. Similarly, the unconventional myosins, Va, VIIa and XV, which can act as effectors for Rab27a in some cell types, are not required in CTL. These results reveal differences in the protein machinery required for biogenesis and/or secretion of lysosome‐related organelles in CTL and melanocytes.