Human Cancers Express TRAILshort, a Dominant Negative TRAIL Splice Variant, Which Impairs Immune Effector Cell Killing of Tumor Cells.

PURPOSE: TNF-related apoptosis inducing ligand (TRAIL) expression by immune cells contributes to antitumor immunity. A naturally occurring splice variant of TRAIL, called TRAILshort, antagonizes TRAIL-dependent cell killing. It is unknown whether tumor cells express TRAILshort and if it impacts antitumor immunity. EXPERIMENTAL DESIGN: We used an unbiased informatics approach to identify TRAILshort expression in primary human cancers, and validated those results with IHC and ISH. TRAILshort-specific mAbs were used to determine the effect of TRAILshort on tumor cell sensitivity to TRAIL, and to immune effector cell dependent killing of autologous primary tumors. RESULTS: As many as 40% of primary human tumors express TRAILshort by both RNA sequencing and IHC analysis. By ISH, TRAILshort expression is present in tumor cells and not bystander cells. TRAILshort inhibition enhances cancer cell lines sensitivity to TRAIL-dependent killing both in vitro and in immunodeficient xenograft mouse models. Immune effector cells isolated from patients with B-cell malignancies killed more autologous tumor cells in the presence compared with the absence of TRAILshort antibody (P < 0.05). CONCLUSIONS: These results identify TRAILshort in primary human malignancies, and suggest that TRAILshort blockade can augment the effector function of autologous immune effector cells.See related commentary by de Miguel and Pardo, p. 5546.

HIV Protease-Generated Casp8p41, When Bound and Inactivated by Bcl2, Is Degraded by the Proteasome.

HIV protease is known to cause cell death, which is dependent upon cleavage of procaspase 8. HIV protease cleavage of procaspase 8 generates Casp8p41, which directly binds Bak with nanomolar affinity, causing Bak activation and consequent cell death. Casp8p41 can also bind Bcl2 with nanomolar affinity, in which case cell death is averted. Central memory CD4 T cells express high levels of Bcl2, possibly explaining why those cells do not die when they reactivate HIV. Here, we determine that the Casp8p41-Bcl2 complex is polyubiquitinated and degraded by the proteasome. Ixazomib, a proteasome inhibitor in clinical use, blocks this pathway, increasing the abundance of Casp8p41 and causing more cells to die in a Casp8p41-dependent manner.IMPORTANCE The Casp8p41 pathway of cell death is unique to HIV-infected cells yet is blocked by Bcl2. Once bound by Bcl2, Casp8p41 is polyubiquitinated and degraded by the proteasome. Proteasome inhibition blocks degradation of Casp8p41, increasing Casp8p41 levels and causing more HIV-infected cells to die.

Both HIV-Infected and Uninfected Cells Express TRAILshort, Which Confers TRAIL Resistance upon Bystander Cells within the Microenvironment.

TNF-related apoptosis-inducing ligand (TRAIL) was initially described to induce apoptosis of tumor cells and/or virally infected cells, although sparing normal cells, and has been implicated in the pathogenesis of HIV disease. We previously identified TRAILshort, a TRAIL splice variant, in HIV-infected patients and characterized it as being a dominant negative ligand to subvert TRAIL-mediated killing. Herein, using single-cell genomics we demonstrate that TRAILshort is produced by HIV-infected cells, as well as by uninfected bystander cells, and that the dominant stimulus which induces TRAILshort production are type I IFNs and TLR7, TLR8, and TLR9 agonists. TRAILshort has a short t1/2 by virtue of containing a PEST domain, which targets the protein toward the ubiquitin proteasome pathway for degradation. Further we show that TRAILshort binds preferentially to TRAIL receptors 1 and 2 with significantly reduced interaction with the decoy TRAIL receptors 3 and 4. Recombinant TRAILshort is sufficient to protect cells against TRAIL-induced killing, whereas immunodepletion of TRAILshort with a specific Ab restores TRAIL sensitivity. Importantly we show that TRAILshort is shed in microvesicles into the cellular microenvironment and therefore confers TRAIL resistance not only on the cell which produces it, but also upon neighboring bystander cells. These results establish a novel paradigm for understanding and overcoming TRAIL resistance, in particular how HIV-infected cells escape immune elimination by the TRAIL:TRAILshort receptor axis.

Conversion of the LIMA1 tumour suppressor into an oncogenic LMO-like protein by API2-MALT1 in MALT lymphoma.

MALT1 is the only known paracaspase and is a critical mediator of B- and T-cell receptor signalling. The function of the MALT1 gene is subverted by oncogenic chimeric fusions arising from the recurrent t(11;18)(q21;q21) aberration, which is the most frequent translocation in mucosa-associated lymphoid tissue (MALT) lymphoma. API2-MALT1-positive MALT lymphomas manifest antibiotic resistance and aggressive clinical behaviour with poor clinical outcome. However, the mechanisms underlying API2-MALT1-induced MALT lymphomagenesis are not fully understood. Here we show that API2-MALT1 induces paracaspase-mediated cleavage of the tumour suppressor protein LIMA1. LIMA1 binding by API2-MALT1 is API2 dependent and proteolytic cleavage is dependent on MALT1 paracaspase activity. Intriguingly, API2-MALT1-mediated proteolysis generates a LIM domain-only (LMO)-containing fragment with oncogenic properties in vitro and in vivo. Importantly, primary MALT lymphomas harbouring the API2-MALT1 fusion uniquely demonstrate LIMA1 cleavage fragments. Our studies reveal a novel paracaspase-mediated oncogenic gain-of-function mechanism in the pathogenesis of MALT lymphoma.

Patients with discordant responses to antiretroviral therapy have impaired killing of HIV-infected T cells.

In medicine, understanding the pathophysiologic basis of exceptional circumstances has led to an enhanced understanding of biology. We have studied the circumstance of HIV-infected patients in whom antiretroviral therapy results in immunologic benefit, despite virologic failure. In such patients, two protease mutations, I54V and V82A, occur more frequently. Expressing HIV protease containing these mutations resulted in less cell death, caspase activation, and nuclear fragmentation than wild type (WT) HIV protease or HIV protease containing other mutations. The impaired induction of cell death was also associated with impaired cleavage of procaspase 8, a requisite event for HIV protease mediated cell death. Primary CD4 T cells expressing I54V or V82A protease underwent less cell death than with WT or other mutant proteases. Human T cells infected with HIV containing these mutations underwent less cell death and less Casp8p41 production than WT or HIV containing other protease mutations, despite similar degrees of viral replication. The reductions in cell death occurred both within infected cells, as well as in uninfected bystander cells. These data indicate that single point mutations within HIV protease which are selected in vivo can significantly impact the ability of HIV to kill CD4 T cells, while not impacting viral replication. Therefore, HIV protease regulates both HIV replication as well as HIV induced T cell depletion, the hallmark of HIV pathogenesis.

HIV Protease Cleavage of Procaspase 8 is Necessary for Death of HIV-Infected Cells.

Numerous host and viral factors are capable of causing death of HIV infected cells, uninfected bystander cells, or both. We assessed the relevance of HIV protease in infected cell killing by mutating its obligate substrate for death, procaspase 8. VSV pseudotyped HIV infection of cells expressing WT caspase 8 resulted in apoptotic cell death and generation of the HIV protease specific cleavage product of procaspase 8, casp8p41. Conversely, both cell death and casp8p41 production were inhibited in cells expressing procaspase 8 engineered to be resistant to HIV protease cleavage. Lymph nodes from HIV-infected patients with ongoing viral replication also selectively expressed casp8p41, which colocalized with both infected and apoptotic cells. HIV protease cleavage of procaspase 8 appears to be a necessary event for infected cell killing, which is responsible for infected cell death within lymphoid tissues from HIV-infected patients.

Human immunodeficiency virus type 1 protease cleaves procaspase 8 in vivo.

Human immunodeficiency virus type 1 (HIV-1) infection causes apoptosis of infected CD4 T cells as well as uninfected (bystander) CD4 and CD8 T cells. It remains unknown what signals cause infected cells to die. We demonstrate that HIV-1 protease specifically cleaves procaspase 8 to create a novel fragment termed casp8p41, which independently induces apoptosis. casp8p41 is specific to HIV-1 protease-induced death but not other caspase 8-dependent death stimuli. In HIV-1-infected patients, casp8p41 is detected only in CD4(+) T cells, predominantly in the CD27(+) memory subset, its presence increases with increasing viral load, and it colocalizes with both infected and apoptotic cells. These data indicate that casp8p41 independently induces apoptosis and is a specific product of HIV-1 protease which may contribute to death of HIV-1-infected cells.

Analysis of HIV Protease Killing Through Caspase 8 Reveals a Novel Interaction Between Caspase 8 and Mitochondria.

Human Immunodeficiency Virus (HIV) protease initiates apoptosis of HIV-infected cells by proteolytic cleavage of procaspase 8, creating a novel peptide termed casp8p41. Expression of casp8p41 alone is sufficient to initiate caspase-dependent cell death associated with mitochondrial depolarization. Since casp8p41 does not contain the catalytic cysteine at position 360, the mechanism by which casp8p41 initiates apoptosis is unclear. We demonstrate that casp8p41 directly causes mitochondrial depolarization and release of cytochrome c with downstream caspase 9 activation. Moreover, death induced by casp8p41 requires the presence of mitochondria, and in intact cells, casp8p41 colocalizes with mitochondria. These results illuminate a novel mechanism of cell death induced by a caspase 8 cleavage fragment whereby mitochondrial interaction leads to depolarization and cytochrome c release.

Ritonavir does not inhibit calpain in vitro.

Ritonavir, an inhibitor of HIV-1 protease, has been reported to also inhibit the Ca2+-dependent cysteine protease, calpain. We have investigated these claims with an in vitro study of the effect of ritonavir on the m-calpain and mu-calpain isoforms. Ritonavir failed to block either autolytic or hydrolytic calpain activity, but remained fully capable of inhibiting the HIV-1 protease. Any calpain-related effects of ritonavir in cells must, therefore, arise by a mechanism other than direct inhibition of calpains.

Differential effects of interleukin-7 and interleukin-15 on NK cell anti-human immunodeficiency virus activity.

The ability of interleukin-7 (IL-7) and IL-15 to expand and/or augment effector cell functions may be of therapeutic benefit to human immunodeficiency virus (HIV)-infected patients. The functional effects of these cytokines on innate HIV-specific immunity and their impact on cells harboring HIV are unknown. We demonstrate that both IL-7 and IL-15 augment natural killer (NK) function by using cells (CD3(-) CD16(+) CD56(+)) from both HIV-positive and -negative donors. Whereas IL-7 enhances NK function through upregulation of Fas ligand, the effect of IL-15 is mediated through upregulation of tumor necrosis factor-related apoptosis-inducing ligand. The difference in these effector mechanisms is reflected by the ability of IL-15-treated but not IL-7-treated NK cells to reduce the burden of replication-competent HIV in autologous peripheral blood mononuclear cells (PBMC) (infectious units per million for control NK cells, 6.79; for IL-7-treated NK cells, 236.17; for IL-15-treated cells, 1.01; P = 0.01 versus control). In addition, the treatment of PBMC with IL-15-treated but not IL-7-treated NK cells causes undetectable HIV p24 (five of five cases), HIV RNA (five of five cases), or HIV DNA (three of five cases). These results support the concept of adjuvant immunotherapy of HIV infection with either IL-7 or IL-15 but suggest that the NK-mediated antiviral effect of IL-15 may be superior.

Vpr R77Q is associated with long-term nonprogressive HIV infection and impaired induction of apoptosis.

The absence of immune defects that occurs in the syndrome of long-term nonprogressive (LTNP) HIV infection offers insights into the pathophysiology of HIV-induced immune disease. The (H[F/S]RIG)(2) domain of viral protein R (Vpr) induces apoptosis and may contribute to HIV-induced T cell depletion. We demonstrate a higher frequency of R77Q Vpr mutations in patients with LTNP than in patients with progressive disease. In addition, T cell infections using vesicular stomatitis virus G (VSV-G) pseudotyped HIV-1 Vpr R77Q result in less (P = 0.01) T cell death than infections using wild-type Vpr, despite similar levels of viral replication. Wild-type Vpr-associated events, including procaspase-8 and -3 cleavage, loss of mitochondrial transmembrane potential (deltapsi(m)), and DNA fragmentation factor activation are attenuated by R77Q Vpr. These data highlight the pathophysiologic role of Vpr in HIV-induced immune disease and suggest a novel mechanism of LTNP.

Osmotic stress-induced remodeling of the cortical cytoskeleton.

Osmotic stress is known to affect the cytoskeleton; however, this adaptive response has remained poorly characterized, and the underlying signaling pathways are unexplored. Here we show that hypertonicity induces submembranous de novo F-actin assembly concomitant with the peripheral translocation and colocalization of cortactin and the actin-related protein 2/3 (Arp2/3) complex, which are key components of the actin nucleation machinery. Additionally, hyperosmolarity promotes the association of cortactin with Arp2/3 as revealed by coimmunoprecipitation. Using various truncation or phosphorylation-incompetent mutants, we show that cortactin translocation requires the Arp2/3- or the F-actin binding domain, but the process is independent of the shrinkage-induced tyrosine phosphorylation of cortactin. Looking for an alternative signaling mechanism, we found that hypertonicity stimulates Rac and Cdc42. This appears to be a key event in the osmotically triggered cytoskeletal reorganization, because 1) constitutively active small GTPases translocate cortactin, 2) Rac and cortactin colocalize at the periphery of hypertonically challenged cells, and 3) dominant-negative Rac and Cdc42 inhibit the hypertonicity-provoked cortactin and Arp3 translocation. The Rho family-dependent cytoskeleton remodeling may be an important osmoprotective response that reinforces the cell cortex.