Asymmetric cell division is a potential means by which cell fate choices during an immune response are orchestrated. Defining the molecular mechanisms that underlie asymmetric division of T cells is paramount for determining the role of this process in the generation of effector and memory T cell subsets. In other cell types, asymmetric cell division is regulated by conserved polarity protein complexes that control the localization of cell fate determinants and spindle orientation during division. We have developed a tractable, in vitro model of naïve CD8+ T cells undergoing initial division while attached to dendritic cells during antigen presentation to investigate whether similar mechanisms might regulate asymmetric division of T cells. Using this system, we show that direct interactions with antigen presenting cells provide the cue for polarization of T cells. Interestingly, the immunological synapse disseminates before division even though the T cells retain contact with the antigen presenting cell. The cue from the antigen presenting cell is translated into polarization of cell fate determinants via the polarity network of the Par3 and Scribble complexes and orientation of the mitotic spindle during division is orchestrated by the Pins/G protein complex. These findings suggest that T cells have selectively adapted a number of evolutionarily conserved mechanisms to generate diversity through asymmetric cell division.

Granzyme B, a protease released from cytotoxic lymphocytes, has been proposed to induce target cell death by cleaving and activating the pro-apoptotic Bcl-2 family member Bid. It has also been proposed that granzyme B can induce target cell death by activating caspases directly, by cleaving caspase substrates, and/or by cleaving several non-caspase substrates. The relative importance of Bid in granzyme B-induced cell death has therefore remained unclear. Here we report that cells isolated from various tissues of Bid-deficient mice were resistant to granzyme B-induced cell death. Consistent with the proposed role of Bid in regulating mitochondrial outer membrane permeabilization, cytochrome c remained in the mitochondria of Bid-deficient cells treated with granzyme B. Unlike wild type cells, Biddeficient cells survived and were then able to proliferate normally, demonstrating the critical role for Bid in mediating granzyme B-induced apoptosis.Granzyme B is a serine protease contained within the granules of cytotoxic lymphocytes (CLs).1 Upon conjugation with their targets, CLs release their granule contents into the synaptic cleft. Granzyme B then enters the target cell by endocytosis and induces apoptotic death via a perforin-dependent mechanism. The importance of CL-mediated killing in the immune response to various pathogens has made it imperative to understand the mechanism of action of granzyme B.Overexpression of the oncogene Bcl-2 renders cells resistant to granzyme B-induced apoptosis (1, 2), and the cells maintain their ability to proliferate (2, 3). Bcl-2 is one of a large family of proteins that regulate mitochondrial outer membrane permeabilization (MOMP) during apoptosis (4). Pro-apoptotic Bcl-2 family members (such as Bid, Bax, and Bak) induce MOMP (5), whereas anti-apoptotic members (e.g. Bcl-2 and Bcl-XL) prevent MOMP (6). Following MOMP, several pro-apoptotic proteins are released from the mitochondrial intermembrane space. In the cytosol, these proteins facilitate the activation of caspases, proteases that orchestrate the death of a cell by apoptosis. One of these proteins, cytochrome c, initiates a complex with dATP, apoptotic protease-activating factor (APAF-1), and pro-caspase-9. This results in the activation of caspase-9, which in turn activates caspase-3 (7). A second protein SMAC/ Diablo that is also released from the mitochondrial intermembrane space displaces inhibitor of apoptosis proteins (IAPs) from caspases, allowing them to become activated by autoprocessing (8, 9).Granzyme B has been reported to induce MOMP by cleaving and activating the pro-apoptotic Bcl-2 family member Bid after residue [10][11][12]. Granzyme B has also been shown to cleave caspase-3 directly when mixed with cytosolic lysates (13, 14); however, in intact cells, granzyme B only appears to be capable of partially processing procaspase-3 to a p20 form that shows little activity in a cellular context (15). SMAC/Diablo, released following MOMP, then displaces the IAPs from the p20 form of caspase-3, allowing auto-proc...

Toll-like receptor (TLR) agonists can trigger broad inflammatory responses that elicit rapid innate immunity and promote the activities of lymphocytes, which can potentially enhance adoptive immunotherapy in the tumor-bearing setting. In the present study, we found that Polyinosinic:Polycytidylic Acid [Poly(I:C)] and CpG oligodeoxynucleotide 1826 [CpG], agonists for TLR 3 and 9, respectively, potently activated adoptively transferred T cells against a murine model of established melanoma. Intratumoral injection of Poly(I:C) and CpG, combined with systemic transfer of activated pmel-1 T cells, specific for gp10025–33, led to enhanced survival and eradication of 9-day established subcutaneous B16F10 melanomas in a proportion of mice. A series of survival studies in knockout mice supported a key mechanistic pathway, whereby TLR agonists acted via host cells to enhance IFN-γ production by adoptively transferred T cells. IFN-γ, in turn, enhanced the immunogenicity of the B16F10 melanoma line, leading to increased killing by adoptively transferred T cells. Thus, this combination approach counteracted tumor escape from immunotherapy via downregulation of immunogenicity. In conclusion, TLR agonists may represent advanced adjuvants within the setting of adoptive T-cell immunotherapy of cancer and hold promise as a safe means of enhancing this approach within the clinic.

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Human GraB (hGraB) preferentially induces apoptosis via Bcl-2-regulated mitochondrial damage but can also directly cleave caspases and caspase substrates in cell-free systems. How hGraB kills cells when it is delivered by cytotoxic lymphocytes (CL) and the contribution of hGraB to CL-induced death is still not clear. We show that primary human natural killer (hNK) cells, which specifically used hGraB to induce target cell death, were able to induce apoptosis of cells whose mitochondria were protected by Bcl-2. Purified hGraB also induced apoptosis of Bcl-2-overexpressing targets but only when delivered at 5-to 10-fold the concentration required to kill cells expressing endogenous Bcl-2. Caspases were critical in this process as inhibition of caspase activity permitted clonogenic survival of Bcl-2-overexpressing cells treated with hGraB or hNK cells but did not protect cells that only expressed endogenous Bcl-2. Our data therefore show that hGraB triggers caspase activation via mitochondria-dependent and mitochondria-independent mechanisms that are activated in a hierarchical manner, and that the combined effects of Bcl-2 and direct caspase inhibition can block cell death induced by hGraB and primary hNK cells.