Frederic Wagner

T Helper 1 and T Helper 2 Cells Are Pathogenic in an Antigen-specific Model of Colitis

Dysregulated T cell responses to enteric bacteria have been implicated as a common mechanism underlying pathogenesis in rodent models of colitis. However, the bacterial species and T cell specificities that induce disease have been poorly defined. We have developed a model system in which target antigen, bacterial host, and corresponding T cell specificity are defined. OVA-specific T cells from DO11.RAG-2−/− TCR transgenic mice were transferred into RAG-2−/− recipients whose intestinal tracts were colonized with OVA-expressing or control Escherichia coli. Transfer of antigen-naive DO11.RAG-2−/− T cells into recipients colonized with OVA-E. coli resulted in enhanced intestinal recruitment and cell cycling of OVA-specific T cells; however, there was no development of disease. In contrast, transfer of polarized T helper (Th) 1 and Th2 populations resulted in severe wasting and colitis in recipients colonized with OVA-expressing but not control E. coli. The histopathologic features of disease induced by Th1 and Th2 transfers were distinct, but disease severity was comparable. Induction of disease by both Th1 and Th2 transfers was dependent on bacterially associated OVA. These results establish that a single bacterially associated antigen can drive the progression of colitis mediated by both Th1 and Th2 cells and provide a new model for understanding the immunoregulatory interactions between T cells responsive to gut floral antigens.

Hit-and-Run Stimulation: a Novel Concept To Reactivate Latent HIV-1 Infection without Cytokine Gene Induction

ABSTRACT Current antiretroviral therapy (ART) efficiently controls HIV-1 replication but fails to eradicate the virus. Even after years of successful ART, HIV-1 can conceal itself in a latent state in long-lived CD4+ memory T cells. From this latent reservoir, HIV-1 rebounds during treatment interruptions. Attempts to therapeutically eradicate this viral reservoir have yielded disappointing results. A major problem with previously utilized activating agents is that at the concentrations required for efficient HIV-1 reactivation, these stimuli trigger high-level cytokine gene expression (hypercytokinemia). Therapeutically relevant HIV-1-reactivating agents will have to trigger HIV-1 reactivation without the induction of cytokine expression. We present here a proof-of-principle study showing that this is a possibility. In a high-throughput screening effort, we identified an HIV-1-reactivating protein factor (HRF) secreted by the nonpathogenic bacterium Massilia timonae. In primary T cells and T-cell lines, HRF triggered a high but nonsustained peak of nuclear factor kappa B (NF-κB) activity. While this short NF-κB peak potently reactivated latent HIV-1 infection, it failed to induce gene expression of several proinflammatory NF-κB-dependent cellular genes, such as those for tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8), and gamma interferon (IFN-γ). Dissociation of cellular and viral gene induction was achievable, as minimum amounts of Tat protein, synthesized following application of a short NF-κB pulse, triggered HIV-1 transactivation and subsequent self-perpetuated HIV-1 expression. In the absence of such a positive feedback mechanism, cellular gene expression was not sustained, suggesting that strategies modulating the NF-κB activity profile could be used to selectively trigger HIV-1 reactivation.

Optimization of HIV-1 infectivity assays

HIV-1 reporter cell lines are the backbone of diagnostic assays, vaccine and drug development efforts. Performing HIV-1 infection experiments in a T cell background is desirable for many reasons. However, a low susceptibility to infection with primary patient isolates in available reporter T cell lines has limited such efforts. We here demonstrate that optimization of HIV-1 receptor expression and the utilization of serum free medium compositions can increase susceptibility of reporter T cell lines to HIV-1 infection by up to two orders of magnitude.

Kinase control of latent HIV-1 infection: PIM-1 kinase as a major contributor to HIV-1 reactivation.

ABSTRACT Despite the clinical relevance of latent HIV-1 infection as a block to HIV-1 eradication, the molecular biology of HIV-1 latency remains incompletely understood. We recently demonstrated the presence of a gatekeeper kinase function that controls latent HIV-1 infection. Using kinase array analysis, we here expand on this finding and demonstrate that the kinase activity profile of latently HIV-1-infected T cells is altered relative to that of uninfected T cells. A ranking of altered kinases generated from these kinome profile data predicted PIM-1 kinase as a key switch involved in HIV-1 latency control. Using genetic and pharmacologic perturbation strategies, we demonstrate that PIM-1 activity is indeed required for HIV-1 reactivation in T cell lines and primary CD4 T cells. The presented results thus confirm that kinases are key contributors to HIV-1 latency control. In addition, through mutational studies we link the inhibitory effect of PIM-1 inhibitor IV (PIMi IV) on HIV-1 reactivation to an AP-1 motif in the CD28-responsive element of the HIV-1 long terminal repeat (LTR). The results expand our conceptual understanding of the dynamic interactions of the host cell and the latent HIV-1 integration event and position kinome profiling as a research tool to reveal novel molecular mechanisms that can eventually be targeted to therapeutically trigger HIV-1 reactivation.

Selected Drugs with Reported Secondary Cell-Differentiating Capacity Prime Latent HIV-1 Infection for Reactivation

ABSTRACT Reactivation of latent HIV-1 infection is considered our best therapeutic means to eliminate the latent HIV-1 reservoir. Past therapeutic attempts to systemically trigger HIV-1 reactivation using single drugs were unsuccessful. We thus sought to identify drug combinations consisting of one component that would lower the HIV-1 reactivation threshold and a synergistic activator. With aclacinomycin and dactinomycin, we initially identified two FDA-approved drugs that primed latent HIV-1 infection in T cell lines and in primary T cells for reactivation and facilitated complete reactivation at the population level. This effect was correlated not with the reported primary drug effects but with the cell-differentiating capacity of the drugs. We thus tested other cell-differentiating drugs/compounds such as cytarabine and aphidicolin and found that they also primed latent HIV-1 infection for reactivation. This finding extends the therapeutic promise of N′-N′-hexamethylene-bisacetamide (HMBA), another cell-differentiating agent that has been reported to trigger HIV-1 reactivation, into the group of FDA-approved drugs. To this end, it is also noteworthy that suberoylanilide hydroxamic acid (SAHA), a polar compound that was initially developed as a second-generation cell-differentiating agent using HMBA as a structural template and which is now marketed as the histone deacetylase (HDAC) inhibitor vorinostat, also has been reported to trigger HIV-1 reactivation. Our findings suggest that drugs with primary or secondary cell-differentiating capacity should be revisited as HIV-1-reactivating agents as some could potentially be repositioned as candidate drugs to be included in an induction therapy to trigger HIV-1 reactivation.

The Quality of Chimpanzee T-Cell Activation and Simian Immunodeficiency Virus/Human Immunodeficiency Virus Susceptibility Achieved via Antibody-Mediated T-Cell Receptor/CD3 Stimulation Is a Function of the Anti-CD3 Antibody Isotype

ABSTRACT While human immunodeficiency virus type 1 (HIV-1) infection is associated with hyperimmune activation and systemic depletion of CD4+ T cells, simian immunodeficiency virus (SIV) infection in sooty mangabeys or chimpanzees does not exhibit these hallmarks. Control of immune activation is thought to be one of the major components that govern species-dependent differences in the disease pathogenesis. A previous study introduced the idea that the resistance of chimpanzees to SIVcpz infection-induced hyperimmune activation could be the result of the expression of select sialic acid-recognizing immunoglobulin (Ig)-like lectin (Siglec) superfamily members by chimpanzee T cells. Siglecs, which are absent on human T cells, were thought to control levels of T-cell activation in chimpanzees and were thus suggested as a cause for the pathogenic differences in the course of SIVcpz or HIV-1 infection. As in human models of T-cell activation, stimulation had been attempted using an anti-CD3 monoclonal antibody (MAb) (UCHT1; isotype IgG1), but despite efficient binding, UCHT1 failed to activate chimpanzee T cells, an activation block that could be partially overcome by MAb-induced Siglec-5 internalization. We herein demonstrate that anti-CD3 MAb-mediated chimpanzee T-cell activation is a function of the anti-CD3 MAb isotype and is not governed by Siglec expression. While IgG1 anti-CD3 MAbs fail to stimulate chimpanzee T cells, IgG2a anti-CD3 MAbs activate chimpanzee T cells in the absence of Siglec manipulations. Our results thus imply that prior to studying possible differences between human and chimpanzee T-cell activation, a relevant model of chimpanzee T cell activation needs to be established.

Protein phosphatase, Mg 2+ /Mn 2+ -dependent 1A controls the innate antiviral and antibacterial response of macrophages during HIV-1 and Mycobacterium tuberculosis infection

Co-infection with HIV-1 and Mycobacterium tuberculosis (Mtb) is a major public health issue. While some research has described how each pathogen accelerates the course of infection of the other pathogen by compromising the immune system, very little is known about the molecular biology of HIV-1/Mtb co-infection at the host cell level. This is somewhat surprising, as both pathogens are known to replicate and persist in macrophages. We here identify Protein Phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A) as a molecular link between Mtb infection and increased HIV-1 susceptibility of macrophages. We demonstrate that both Mtb and HIV-1 infection induce the expression of PPM1A in primary human monocyte/macrophages and THP-1 cells. Genetic manipulation studies revealed that increased PPMA1 expression rendered THP-1 cells highly susceptible to HIV-1 infection, while depletion of PPM1A rendered them relatively resistant to HIV-1 infection. At the same time, increased PPM1A expression abrogated the ability of THP-1 cells to respond to relevant bacterial stimuli with a proper cytokine/chemokine secretion response, blocked their chemotactic response and impaired their ability to phagocytose bacteria. These data suggest that PPM1A, which had previously been shown to play a role in the antiviral response to Herpes Simplex virus infection, also governs the antibacterial response of macrophages to bacteria, or at least to Mtb infection. PPM1A thus seems to play a central role in the innate immune response of macrophages, implying that host directed therapies targeting PPM1A could be highly beneficial, in particular for HIV/Mtb co-infected patients.

Stable Phenotypic Changes of the Host T Cells are Essential to the Long-term Stability of Latent HIV-1 Infection

ABSTRACT The extreme stability of the latent HIV-1 reservoir in the CD4+ memory T cell population prevents viral eradication with current antiretroviral therapy. It has been demonstrated that homeostatic T cell proliferation and clonal expansion of latently infected T cells due to viral integration into specific genes contribute to this extraordinary reservoir stability. Nevertheless, given the constant exposure of the memory T cell population to specific antigen or bystander activation, this reservoir stability seems remarkable, unless it is assumed that latent HIV-1 resides exclusively in memory T cells that recognize rare antigens. Another explanation for the stability of the reservoir could be that the latent HIV-1 reservoir is associated with an unresponsive T cell phenotype. We demonstrate here that host cells of latent HIV-1 infection events were functionally altered in ways that are consistent with the idea of an anergic, unresponsive T cell phenotype. Manipulations that induced or mimicked an anergic T cell state promoted latent HIV-1 infection. Kinome analysis data reflected this altered host cell phenotype at a system-wide level and revealed how the stable kinase activity changes networked to stabilize latent HIV-1 infection. Protein-protein interaction networks generated from kinome data could further be used to guide targeted genetic or pharmacological manipulations that alter the stability of latent HIV-1 infection. In summary, our data demonstrate that stable changes to the signal transduction and transcription factor network of latently HIV-1 infected host cells are essential to the ability of HIV-1 to establish and maintain latent HIV-1 infection status. IMPORTANCE The extreme stability of the latent HIV-1 reservoir allows the infection to persist for the lifetime of a patient, despite completely suppressive antiretroviral therapy. This extreme reservoir stability is somewhat surprising, since the latently HIV-1 infected CD4+ memory T cells that form the structural basis of the viral reservoir should be exposed to cognate antigen over time. Antigen exposure would trigger a recall response and should deplete the reservoir, likely over a relatively short period. Our data demonstrate that stable and system-wide phenotypic changes to host cells are a prerequisite for the establishment and maintenance of latent HIV-1 infection events. The changes observed are consistent with an unresponsive, anergy-like T cell phenotype of latently HIV-1 infected host cells. An anergy-like, unresponsive state of the host cells of latent HIV-1 infection events would explain the stability of the HIV-1 reservoir in the face of continuous antigen exposure.

Mycobacterium tuberculosis exploits the PPM1A signaling pathway to block host macrophage apoptosis

The ability to suppress host macrophage apoptosis is essential for M. tuberculosis (Mtb) to replicate intracellularly while protecting it from antibiotic treatment. We recently described that Mtb infection upregulated expression of the host phosphatase PPM1A, which impairs the antibacterial response of macrophages. Here we establish PPM1A as a checkpoint target used by Mtb to suppress macrophage apoptosis. Overproduction of PPM1A suppressed apoptosis of Mtb-infected macrophages by a mechanism that involves inactivation of the c-Jun N-terminal kinase (JNK). Targeted depletion of PPM1A by shRNA or inhibition of PPM1A activity by sanguinarine restored JNK activation, resulting in increased apoptosis of Mtb-infected macrophages. We also demonstrate that activation of JNK by subtoxic concentrations of anisomycin induced selective apoptotic killing of Mtb-infected human macrophages, which was completely blocked in the presence of a specific JNK inhibitor. Finally, selective killing of Mtb-infected macrophages and subsequent bacterial release enabled rifampicin to effectively kill Mtb at concentrations that were insufficient to act against intracellular Mtb, providing proof of principle for the efficacy of a “release and kill” strategy. Taken together, these findings suggest that drug-induced selective apoptosis of Mtb-infected macrophages is achievable.

CD151 Expression Is Associated with a Hyperproliferative T Cell Phenotype

The tetraspanin CD151 is a marker of aggressive cell proliferation and invasiveness for a variety of cancer types. Given reports of CD151 expression on T cells, we explored whether CD151 would mark T cells in a hyperactivated state. Consistent with the idea that CD151 could mark a phenotypically distinct T cell subset, it was not uniformly expressed on T cells. CD151 expression frequency was a function of the T cell lineage (CD8 > CD4) and a function of the memory differentiation state (naive T cells < central memory T cells < effector memory T cells < T effector memory RA+ cells). CD151 and CD57, a senescence marker, defined the same CD28− T cell populations. However, CD151 also marked a substantial CD28+ T cell population that was not marked by CD57. Kinome array analysis demonstrated that CD28+CD151+ T cells form a subpopulation with a distinct molecular baseline and activation phenotype. Network analysis of these data revealed that cell cycle control and cell death were the most altered process motifs in CD28+CD151+ T cells. We demonstrate that CD151 in T cells is not a passive marker, but actively changed the cell cycle control and cell death process motifs of T cells. Consistent with these data, long-term T cell culture experiments in the presence of only IL-2 demonstrated that independent of their CD28 expression status, CD151+ T cells, but not CD151− T cells, would exhibit an Ag-independent, hyperresponsive proliferation phenotype. Not unlike its reported function as a tumor aggressiveness marker, CD151 in humans thus marks and enables hyperproliferative T cells.