Optimizing single cell proteomics using trapped ion mobility spectrometry for label-free experiments.

Although single cell RNA-seq has had a tremendous impact on biological research, a corresponding technology for unbiased mass spectrometric analysis of single cells has only recently become available. Significant technological breakthroughs including miniaturized sample handling have enabled proteome profiling of single cells. Furthermore, trapped ion mobility spectrometry (TIMS) in combination with parallel accumulation-serial fragmentation operated in data-dependent acquisition mode (DDA-PASEF) allowed improved proteome coverage from low-input samples. It has been demonstrated that modulating the ion flux in TIMS affects the overall performance of proteome profiling. However, the effect of TIMS settings on the analysis of low-input samples has been less investigated. Thus, we sought to optimize the conditions of TIMS with regard to ion accumulation/ramp times and ion mobility range for low-input samples. We observed that an ion accumulation time of 180 ms and monitoring a narrower ion mobility range from 0.7 to 1.3 V s cm-2 resulted in a substantial gain in the depth of proteome coverage and in detecting proteins with low abundance. We used these optimized conditions for proteome profiling of sorted human primary T cells, which yielded an average of 365, 804, 1116, and 1651 proteins from single, five, ten, and forty T cells, respectively. Notably, we demonstrated that the depth of proteome coverage from a low number of cells was sufficient to delineate several essential metabolic pathways and the T cell receptor signaling pathway. Finally, we showed the feasibility of detecting post-translational modifications including phosphorylation and acetylation from single cells. We believe that such an approach could be applied to label-free analysis of single cells obtained from clinically relevant samples.

The BCL-2 Inhibitor Venetoclax Augments Immune Effector Function Mediated by Fas Ligand, TRAIL, and Perforin/Granzyme B, Resulting in Reduced Plasma Viremia and Decreased HIV Reservoir Size during Acute HIV Infection in a Humanized Mouse Model.

The BCL-2 prosurvival protein is implicated in HIV persistence and is a potential therapeutic target for HIV eradication efforts. We now know that cells harboring HIV are preferentially enriched for high BCL-2 expression, enabling their survival, and that the BCL-2 inhibitor venetoclax promotes the death of actively replicating HIV-infected cells in vitro and ex vivo. Herein, we assess the effect of venetoclax on immune clearance of infected cells and show that BCL-2 inhibition significantly enhances target cell killing induced by Fas ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), and perforin/granzyme B and synergistically enhances autologous NK (natural killer) and CD8 cells' killing of target cells. In a humanized mouse model of acute HIV infection, venetoclax monotherapy significantly decreases plasma viremia and normalizes CD4:CD8 ratios, and results in more mice with undetectable provirus levels than control. In this model, treatment was associated with leukopenia, as has been described clinically in patients receiving venetoclax for other indications. These data confirm meaningful anti-HIV effects of venetoclax during HIV infection but suggest that venetoclax use should be combined with ART (antiretroviral therapy) to reduce toxicity. IMPORTANCE This study is the first to examine the applicability of BCL-2 inhibition in the setting of active HIV infection in vivo. Furthermore, this study demonstrates that venetoclax significantly enhances target cell killing induced by Fas ligand, TRAIL, and perforin/granzyme B and synergistically enhances autologous NK and CD8 cells' killing of target cells.

Single center, open label dose escalating trial evaluating once weekly oral ixazomib in ART-suppressed, HIV positive adults and effects on HIV reservoir size in vivo.

BACKGROUND: Achieving a functional or sterilizing cure for HIV will require identification of therapeutic interventions that reduce HIV reservoir size in infected individuals. Proteasome inhibitors, such as ixazomib, impact multiple aspects of HIV biology including latency, transcription initiation, viral replication, and infected cell killing through the HIV protease - Casp8p41 pathway, resulting in latency reversal and reduced measures of HIV reservoir size ex vivo. METHODS: We conducted a phase 1b/2a dose escalating, open label trial of weekly oral ixazomib for 24 weeks in antiretroviral (ART)-suppressed, HIV positive adults (NCT02946047). The study was conducted from March 2017 to August 2019 at two tertiary referral centers in the United States. The primary outcomes were safety and tolerability of oral ixazomib. Secondary outcomes included changes in immunologic markers and estimates of HIV reservoir size after ixazomib treatment. FINDINGS: Sixteen participants completed the study. Ixazomib up to 4mg weekly was safe and well-tolerated, yielding no treatment-emergent events above grade 1. In exploratory analyses, ixazomib treatment was associated with detectable viremia that was below the lower limit of quantification (LLQ) in 9 participants, and viremia that was above LLQ in 4 of 16 participants. While treatment was associated with reduced CD4 counts [baseline 783 cells/ mm3 vs. week-24 724 cells/ mm3 p=0.003], there were no changes in markers of cellular activation, exhaustion or inflammation. Total HIV DNA and proviral sequencing were not altered by ixazomib treatment. Intact proviral DNA assay (IPDA) identified intact proviruses in 14 patients pre-treatment, and in 10/14 of those subjects post treatment values were reduced (P=0.068), allowing a calculated intact proviral half life of 0.6 years (95% CI 0.3, 2.5), compared to 7.1 years (95% CI 3.9, 18, p=0.004) in historical controls. Differentiation Quantitative Viral Outgrowth Assays (dQVOA) identified measurable proviruses in 15 subjects pre-treatment; post-treatment values were numerically reduced in 9, but overall differences were not significantly different. INTERPRETATION: Our study successfully met its primary endpoint of demonstrating the safety of ixazomib for 24 weeks in HIV infected persons. Exploratory analyses suggest that the effects observed ex vivo of latency reversal and reductions in HIV reservoir size, also occur in vivo. Future controlled studies of ixazomib are warranted. FUNDING: This study was funded by Millennium Pharmaceuticals Inc..; the Mayo Clinic Foundation; the National Institutes of Health, including the National Institute of Allergy and Infectious Diseases, Division of AIDS, the National Heart, Lung and Blood Institute, the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute of Neurological Disorders and Stroke, and the National Institute on Drug Abuse. Mayo Clinic also acknowledges generous funding support from Mr. Joseph T. and Mrs. Michele P. Betten.

The Combination of Venetoclax and Ixazomib Selectively and Efficiently Kills HIV-Infected Cell Lines but Has Unacceptable Toxicity in Primary Cell Models.

The antiapoptotic protein BCL2 inhibits death of HIV-infected cells. Previously, we showed that the BCL2 inhibitor venetoclax selectively kills acutely HIV-infected cells and reduces HIV DNA in latently infected CD4 T cells ex vivo after reactivation with anti-CD3/anti-CD28. However, there is a need to identify a combination therapy with venetoclax and a clinically relevant latency reversal agent. Ixazomib is an oral proteasome inhibitor which we have shown reactivates latent HIV and predisposes reactivated cells to cell death. Here, we determined that the combination of venetoclax and ixazomib kills more latently HIV-infected cells and leads to greater reduction in HIV replication than either treatment alone in vitro in a T cell model. However, combination treatment of ex vivo CD4 T cells from antiretroviral therapy (ART)-suppressed, HIV-positive participants resulted in unanticipated and unacceptable nonspecific toxicity in primary cells. Therefore, while we show proof of concept that multiple agents can enhance selective killing of HIV-infected cells, the combination of venetoclax and ixazomib has unacceptable toxicity in primary cells, and so further investigation is needed to identify a clinically relevant latency reversal agent to combine with venetoclax as a novel strategy to reduce the size of the HIV reservoir.IMPORTANCE A cure for HIV would require eliminating cells that contain the virus in a latent form from the body. Current antiretroviral medications are unable to rid the body of latently infected cells. Here, we show that a combination of investigational agents-ixazomib plus venetoclax-which reactivate latent virus and predispose infected cells to apoptosis may reduce latent virus in a T cell model, but at the expense of nonspecific toxicity in primary cells.

Reactivating latent HIV with PKC agonists induces resistance to apoptosis and is associated with phosphorylation and activation of BCL2.

Eradication of HIV-1 by the "kick and kill" strategy requires reactivation of latent virus to cause death of infected cells by either HIV-induced or immune-mediated apoptosis. To date this strategy has been unsuccessful, possibly due to insufficient cell death in reactivated cells to effectively reduce HIV-1 reservoir size. As a possible cause for this cell death resistance, we examined whether leading latency reversal agents (LRAs) affected apoptosis sensitivity of CD4 T cells. Multiple LRAs of different classes inhibited apoptosis in CD4 T cells. Protein kinase C (PKC) agonists bryostatin-1 and prostratin induced phosphorylation and enhanced neutralizing capability of the anti-apoptotic protein BCL2 in a PKC-dependent manner, leading to resistance to apoptosis induced by both intrinsic and extrinsic death stimuli. Furthermore, HIV-1 producing CD4 T cells expressed more BCL2 than uninfected cells, both in vivo and after ex vivo reactivation. Therefore, activation of BCL2 likely contributes to HIV-1 persistence after latency reversal with PKC agonists. The effects of LRAs on apoptosis sensitivity should be considered in designing HIV cure strategies predicated upon the "kick and kill" paradigm.

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.

TRAILshort Protects against CD4 T Cell Death during Acute HIV Infection.

CD4 T cells from HIV-1 infected patients die at excessive rates compared to those from uninfected patients, causing immunodeficiency. We previously identified a dominant negative ligand that antagonizes the TRAIL-dependent pathway of cell death, which we called TRAILshort. Because the TRAIL pathway has been implicated in CD4 T cell death occurring during HIV-1 infection, we used short hairpin RNA knockdown, CRISPR deletion, or Abs specific for TRAILshort to determine the effect of inhibiting TRAILshort on the outcome of experimental acute HIV infection in vitro. Strikingly, all three approaches to TRAILshort deletion/inhibition enhanced HIV-induced death of both infected and uninfected human CD4 T cells. Thus, TRAILshort impacts T cell dynamics during HIV infection, and inhibiting TRAILshort causes more HIV-infected and uninfected bystander cells to die. TRAILshort is, therefore, a host-derived, host-adaptive mechanism to limit the effects of TRAIL-induced cell death. Further studies on the effects of TRAILshort in other disease states are warranted.

HIV elite control is associated with reduced TRAILshort expression.

OBJECTIVE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) dependent apoptosis has been implicated in CD4 T-cell death and immunologic control of HIV-1 infection. We have described a splice variant called TRAILshort, which is a dominant negative ligand that antagonizes TRAIL-induced cell death in the context of HIV-1 infection. HIV-1 elite controllers naturally control viral replication for largely unknown reasons. Since enhanced death of infected cells might be responsible, as might occur in situations of low (or inhibited) TRAILshort, we tested whether there was an association between elite controller status and reduced levels of TRAILshort expression. DESIGN: Cohort study comparing TRAILshort and full length TRAIL expression between HIV-1 elite controllers and viremic progressors from two independent populations. METHODS: TRAILshort and TRAIL gene expression in peripheral blood mononuclear cells (PBMCs) was determined by RNA-seq. TRAILshort and TRAIL protein expression in plasma was determined by antibody bead array and proximity extension assay respectively. RESULTS: HIV-1 elite controllers expressed less TRAILshort transcripts in PBMCs (P = 0.002) and less TRAILshort protein in plasma (P < 0.001) than viremic progressors. CONCLUSION: Reduced TRAILshort expression in PBMCs and plasma is associated with HIV-1 elite controller status.

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.

An advanced BLT-humanized mouse model for extended HIV-1 cure studies.

OBJECTIVE: Although bone marrow, liver, thymus (BLT)-humanized mice provide a robust model for HIV-1 infection and enable evaluation of cure strategies dependent on endogenous immune responses, most mice develop graft versus host disease (GVHD), limiting their utility for extended HIV cure studies. This study aimed to: evaluate the GVHD-resistant C57 black 6 (C57BL/6) recombination activating gene 2 (Rag2)γcCD47 triple knockout (TKO)-BLT mouse as a model to establish HIV-1 latency. Determine whether TKO-BLT mice could be maintained on antiretroviral therapy (ART) for extended periods of time. Assess the rapidity of viral rebound following therapy interruption. DESIGN: TKO-BLT mice were HIV-1 infected, treated with various ART regimens over extended periods of time and assayed for viral rebound following therapy interruption. METHODS: Daily subcutaneous injection and oral ART-mediated suppression of HIV-1 infection was tested at various doses in TKO-BLT mice. Mice were monitored for suppression of viremia and cellular HIV-1 RNA and DNA prior to and following therapy interruption. RESULTS: Mice remained healthy for 45 weeks posthumanization and could be treated with ART for up to 18 weeks. Viremia was suppressed to less than 200 copies/ml in the majority of mice with significant reductions in cellular HIV-1 RNA and DNA. Treatment interruption resulted in rapid viral recrudescence. CONCLUSION: HIV-1 latency can be maintained in TKO-BLT mice over extended periods on ART and rapid viral rebound occurs following therapy removal. The additional 15-18 weeks of healthy longevity compared with other BLT models provides sufficient time to examine the decay kinetics of the latent reservoir as well as observe delays in recrudescence in HIV-1 cure studies.

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.

Extensive virologic and immunologic characterization in an HIV-infected individual following allogeneic stem cell transplant and analytic cessation of antiretroviral therapy: A case study.

BACKGROUND: Notwithstanding 1 documented case of HIV-1 cure following allogeneic stem cell transplantation (allo-SCT), several subsequent cases of allo-SCT in HIV-1 positive individuals have failed to cure HIV-1 infection. The aim of our study was to describe changes in the HIV reservoir in a single chronically HIV-infected patient on suppressive antiretroviral therapy who underwent allo-SCT for treatment of acute lymphoblastic leukemia. METHODS AND FINDINGS: We prospectively collected peripheral blood mononuclear cells (PBMCs) by leukapheresis from a 55-year-old man with chronic HIV infection before and after allo-SCT to measure the size of the HIV-1 reservoir and characterize viral phylogeny and phenotypic changes in immune cells. At day 784 post-transplant, when HIV-1 was undetectable by multiple measures-including PCR measurements of both total and integrated HIV-1 DNA, replication-competent virus measurement by large cell input quantitative viral outgrowth assay, and in situ hybridization of colon tissue-the patient consented to an analytic treatment interruption (ATI) with frequent clinical monitoring. He remained aviremic off antiretroviral therapy until ATI day 288, when a low-level virus rebound of 60 HIV-1 copies/ml occurred, which increased to 1,640 HIV-1 copies/ml 5 days later, prompting reinitiation of ART. Rebounding plasma HIV-1 sequences were phylogenetically distinct from proviral HIV-1 DNA detected in circulating PBMCs before transplantation. The main limitations of this study are the insensitivity of reservoir measurements, and the fact that it describes a single case. CONCLUSIONS: allo-SCT led to a significant reduction in the size of the HIV-1 reservoir and a >9-month-long ART-free remission from HIV-1 replication. Phylogenetic analyses suggest that the origin of rebound virus was distinct from the viruses identified pre-transplant in the PBMCs.

Increasing procaspase 8 expression using repurposed drugs to induce HIV infected cell death in ex vivo patient cells.

HIV persists because a reservoir of latently infected CD4 T cells do not express viral proteins and are indistinguishable from uninfected cells. One approach to HIV cure suggests that reactivating HIV will activate cytotoxic pathways; yet when tested in vivo, reactivating cells do not die sufficiently to reduce cell-associated HIV DNA levels. We recently showed that following reactivation from latency, HIV infected cells generate the HIV specific cytotoxic protein Casp8p41 which is produced by HIV protease cleaving procaspase 8. However, cell death is prevented, possibly due to low procaspase 8 expression. Here, we tested whether increasing procaspase 8 levels in CD4 T cells will produce more Casp8p41 following HIV reactivation, causing more reactivated cells to die. Screening 1277 FDA approved drugs identified 168 that increased procaspase 8 expression by at least 1.7-fold. Of these 30 were tested for anti-HIV effects in an acute HIVIIIb infection model, and 9 drugs at physiologic relevant levels significantly reduced cell-associated HIV DNA. Primary CD4 T cells from ART suppressed HIV patients were treated with one of these 9 drugs and reactivated with αCD3/αCD28. Four drugs significantly increased Casp8p41 levels following HIV reactivation, and decreased total cell associated HIV DNA levels (flurbiprofen: p = 0.014; doxycycline: p = 0.044; indomethacin: p = 0.025; bezafibrate: P = 0.018) without effecting the viability of uninfected cells. Thus procaspase 8 levels can be increased pharmacologically and, in the context of HIV reactivation, increase Casp8p41 causing death of reactivating cells and decreased HIV DNA levels. Future studies will be required to define the clinical utility of this or similar approaches.

Maintenance of the HIV Reservoir Is Antagonized by Selective BCL2 Inhibition.

Decay of the HIV reservoir is slowed over time in part by expansion of the pool of HIV-infected cells. This expansion reflects homeostatic proliferation of infected cells by interleukin-7 (IL-7) or antigenic stimulation, as well as new rounds of infection of susceptible target cells. As novel therapies are being developed to accelerate the decay of the latent HIV reservoir, it will be important to identify interventions that prevent expansion and/or repopulation of the latent HIV reservoir. Our previous studies showed that HIV protease cleaves the host protein procaspase 8 to generate Casp8p41, which can bind and activate Bak to induce apoptosis of infected cells. In circumstances where expression of the anti-apoptotic protein BCL2 is high, Casp8p41 instead binds BCL2, and cell death does not occur. This effect can be overcome by treating cells with the clinically approved BCL2 antagonist venetoclax, which prevents Casp8p41 from binding BCL2, thereby allowing Casp8p41 to bind Bak and kill the infected cell. Here we assess whether the events that maintain the HIV reservoir are also antagonized by venetoclax. Using the J-Lat 10.6 model of persistent infection, we demonstrate that proliferation and HIV expression are countered by the use of venetoclax, which causes preferential killing of the HIV-expressing cells. Similarly, during new rounds of infection of primary CD4 T cells, venetoclax causes selective killing of HIV-infected cells, resulting in decreased numbers of HIV DNA-containing cells.IMPORTANCE Cure of HIV infection requires an intervention that reduces the HIV reservoir size. A variety of approaches are being tested for their ability to impact HIV reservoir size. Even if successful, however, these approaches will need to be combined with additional complementary approaches that prevent replenishment or repopulation of the HIV reservoir. Our previous studies have shown that the FDA-approved BCL2 antagonist venetoclax has a beneficial effect on the HIV reservoir size following HIV reactivation. Here we demonstrate that venetoclax also has a beneficial effect on HIV reservoir size in a model of homeostatic proliferation of HIV as well as in acute spreading infection of HIV in primary CD4 T cells. These results suggest that venetoclax, either alone or in combination with other approaches to reducing HIV reservoir size, is a compound worthy of further study for its effects on HIV reservoir size.

Prime, Shock, and Kill: Priming CD4 T Cells from HIV Patients with a BCL-2 Antagonist before HIV Reactivation Reduces HIV Reservoir Size.

UNLABELLED: Understanding how some HIV-infected cells resist the cytotoxicity of HIV replication is crucial to enabling HIV cure efforts. HIV killing of CD4 T cells that replicate HIV can involve HIV protease-mediated cleavage of procaspase 8 to generate a fragment (Casp8p41) that directly binds and activates the mitochondrial proapoptotic protein BAK. Here, we demonstrate that Casp8p41 also binds with nanomolar affinity to the antiapoptotic protein Bcl-2, which sequesters Casp8p41 and prevents apoptosis. Further, we show that central memory CD4 T cells (TCM) from HIV-infected individuals have heightened expression of BCL-2 relative to procaspase 8, possibly explaining the persistence of HIV-infected TCMdespite generation of Casp8p41. Consistent with this hypothesis, the selective BCL-2 antagonist venetoclax induced minimal killing of uninfected CD4 T cells but markedly increased the death of CD4 T cells and diminished cell-associated HIV DNA when CD4 T cells from antiretroviral therapy (ART)-suppressed HIV patients were induced with αCD3/αCD28 to reactivate HIVex vivo Thus, priming CD4 T cells from ART suppressed HIV patients with a BCL-2 antagonist, followed by HIV reactivation, achieves reductions in cell-associated HIV DNA, whereas HIV reactivation alone does not. IMPORTANCE: HIV infection is incurable due to a long-lived reservoir of HIV(+)memory CD4 T cells, and no clinically relevant interventions have been identified that reduce the number of these HIV DNA-containing cells. Since postintegration HIV replication can result in HIV protease generation of Casp8p41, which activates BAK, causing infected CD4 T cell death, we sought to determine whether this occurs in memory CD4 T cells. Here, we demonstrate that memory CD4 T cells can generate Casp8p41 and yet are intrinsically resistant to death induced by diverse stimuli, including Casp8p41. Furthermore, BCL-2 expression is relatively increased in these cells and directly binds and inhibits Casp8p41's proapoptotic effects. Antagonizing BCL-2 with venetoclax derepresses this antagonism, resulting in death, preferentially in HIV DNA containing cells, since only these cells generate Casp8p41. Thus, BCL-2 antagonism is a clinically relevant intervention with the potential to reduce HIV reservoir size in patients.

Casp8p41 generated by HIV protease kills CD4 T cells through direct Bak activation.

Previous studies have shown that human immunodeficiency virus (HIV) protease cleaves procaspase 8 to a fragment, termed Casp8p41, that lacks caspase activity but nonetheless contributes to T cell apoptosis. Herein, we show that Casp8p41 contains a domain that interacts with the BH3-binding groove of pro-apoptotic Bak to cause Bak oligomerization, Bak-mediated membrane permeabilization, and cell death. Levels of active Bak are higher in HIV-infected T cells that express Casp8p41. Conversely, targeted mutations in the Bak-interacting domain diminish Bak binding and Casp8p41-mediated cell death. Similar mutations in procaspase 8 impair the ability of HIV to kill infected T cells. These observations support a novel paradigm in which HIV converts a normal cellular constituent into a direct activator that functions like a BH3-only protein.

Choice of antiretroviral therapy differentially impacts survival of HIV-infected CD4 T cells.

BACKGROUND: HIV eradication strategies are now being evaluated in vitro and in vivo. A cornerstone of such approaches is maximal suppression of viral replication with combination antiretroviral therapy (ART). Since many antiretroviral agents have off target effects, and different classes target different components of the viral life cycle, we questioned whether different classes of ART might differentially affect the survival and persistence of productively HIV-infected CD4 T cells. METHODS: In vitro infections of primary CD4 T cells using clinical isolates of HIV-1 that were either protease inhibitor susceptible (HIV PI-S), or resistant (HIV PI-R) were treated with nothing, lopinavir, efavirenz or raltegravir. Cell viability, apoptosis, and the proportion of surviving cells that were P24 positive was assessed by flow cytometry. RESULTS: In HIV PI-S infected primary cultures, all three antiretroviral agents decreased viral replication, and reduced the total number of cells that were undergoing apoptosis (P < 0.01) similarly. Similarly, in the HIV PI-R infected cultures, both efavirenz and raltegravir reduced viral replication and reduced apoptosis compared to untreated control (P < 0.01), while lopinavir did not, suggesting that HIV replication drives T cell apoptosis, which was confirmed by association by linear regression (P < 0.0001) . However since HIV protease has been suggested to directly induce apoptosis of infected CD4 T cells, and HIV PI are intrinsically antiapoptotic, we evaluated apoptosis in productively infected (HIV P24+) cells. More HIV p24 positive cells were apoptotic in the Efavirenz or raltegravir treated cultures than the lopinavir treated cultures (P = 0.0008 for HIV PI-R and P = 0.06 for the HIV PI-S), indicating that drug class impacts survival of productively infected CD4 T cells. CONCLUSIONS: Inhibiting HIV replication with a PI, NNRTI or INSTI reduces total HIV-induced T cell apoptosis. However, blocking HIV replication with PI but not with NNRTI or INSTI promotes survival of productively HIV-infected cells. Thus, selection of antiretroviral agents may impact the success of HIV eradication strategies.

TRAIL dependent fratricidal killing of gp120 primed hepatocytes by HCV core expressing hepatocytes.

The mechanism by which HIV and HCV cooperatively accelerate hepatocyte damage is not clearly understood; however, each virus affects the TRAIL: TRAIL-receptor system. We, therefore, questioned whether the independent effects of HCV and HIV combine to synergistically result in TRAIL dependent hepatocyte killing. We describe that Huh7 hepatocytes treated with HIV gp120 results in both increase TRAIL-R2 expression and an acquired sensitivity to TRAIL mediated killing. Moreover HCV infection and HCV core expression alone in Huh7 cells upregulates TRAIL. Co-incubation of HIV gp120 primed hepatocytes with HCV core expressing hepatocytes results in the selective death of the HIV gp120 primed hepatocytes that is selectively blocked by TRAIL-R2-Fc fusion protein. Liver biopsies from HIV mono-infected patients have increased TRAIL-R2; biopsies from HCV infected patients have increased TRAIL, while co-infected liver biopsies have increased PARP cleavage within hepatocytes indicating enhanced apoptosis. These findings suggest a pathogenic model to understand why HIV/HCV co-infection accelerates liver injury.

Isolation of a TRAIL antagonist from the serum of HIV-infected patients.

Virus-host interactions are characterized by the selection of adaptive mechanisms by which to evade pathogenic and defense mechanisms, respectively. In primary T cells infected with HIV, HIV infection up-regulates TNF-related apoptosis inducing ligand (TRAIL) and death-inducing TRAIL receptors, but blockade of TRAIL:TRAIL receptor interaction does not alter HIV-induced cell death. Instead, HIV infection results in a novel splice variant that we call TRAIL-short (TRAIL-s), which antagonizes TRAIL-R2. In HIV patients, plasma TRAIL-s concentration increases with increasing viral load and renders cells resistant to TRAIL-induced death. Knockdown of TRAIL-s abrogates this resistance. We propose that TRAIL-s is a novel adaptive mechanism of apoptosis resistance acquired by HIV-infected cells to avoid their elimination by TRAIL-dependent effector mechanism.