Louis Gonzalez

Therapeutic Treg expansion in mice by TNFRSF25 prevents allergic lung inflammation

TNF receptor superfamily member 25 (TNFRSF25; also known as DR3, and referred to herein as TNFR25) is constitutively and highly expressed by CD4(+)FoxP3(+) Tregs. However, its function on these cells has not been determined. Here we used a TNFR25-specific agonistic monoclonal antibody, 4C12, to study the effects of TNFR25 signaling on Tregs in vivo in mice. Signaling through TNFR25 induced rapid and selective expansion of preexisting Tregs in vivo such that they became 30%-35% of all CD4(+) T cells in the peripheral blood within 4 days. TNFR25-induced Treg proliferation was dependent upon TCR engagement with MHC class II, IL-2 receptor, and Akt signaling, but not upon costimulation by CD80 or CD86; it was unaffected by rapamycin. TNFR25-expanded Tregs remained highly suppressive ex vivo, and Tregs expanded by TNFR25 in vivo were protective against allergic lung inflammation, a mouse model for asthma, by reversing the ratio of effector T cells to Tregs in the lung, suppressing IL-13 and Th2 cytokine production, and blocking eosinophil exudation into bronchoalveolar fluid. Our studies define what we believe to be a novel mechanism for Treg control and important functions for TNFR25 in regulating autoaggression that balance its known role in enhancing autoimmunity.

Human immunodeficiency virus type 1 RNA Levels in different regions of human brain: Quantification using real-time reverse transcriptase-polymerase chain reaction

Human immunodeficiency virus type 1 (HIV-1) enters the central nervous system shortly after the infection and becomes localized in different regions of the brain, leading to various neurological abnormalities including motor disorders and neurocognitive deficits. Although HIV-1-associated functional abnormalities of the central nervous system (CNS) can be evaluated during life by using various test batteries, HIV-1 virus concentration in different brain regions can be measured only after death. The tissues obtained at autopsy provide a valuable source for determining the role of various factors, including that of HIV-1 viral load in the CNS, that may contribute to the regional CNS neuropathogenesis. For this study, we obtained from the National Institutes of Health-sponsored National NeuroAIDS Tissue Consortium (NNTC) the tissues from different brain regions collected at autopsy of HIV-1-positive (N = 38) and HIV-negative (N = 11) individuals, with postmortem intervals of 2 to 29 h, and measured HIV-1 RNA concentration in the frontal cortex, frontal cortex area 4, frontal cortex area 6, basal ganglia, caudate nucleus, putamen, globus pallidus, substantia nigra, and cerebrospinal fluid. Because HIV-1+ individuals were infected with the virus for up to 21 years and the majority of them had used highly active antiretroviral therapy (HAART), we used highly sensitive real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay in order to detect a wide dynamic range of HIV-1 RNA with lower detection limit of a single copy. The primers and probes were from the long terminal repeat (LTR) region of HIV genome for achieving higher specificity and sensitivity of detection and amplification. Our results demonstrate a wide variation in the concentration of HIV-1 RNA in different brain regions (5.51 and 8,144,073; log10 0.74 and 6.91 copies/g tissue), and despite the high specificity and sensitivity of this method, viral RNA was not detected in 50% of all the samples, and in 30% to 64% of samples of each region of HIV-1+ individuals. However, the highest concentration of viral RNA was found in the caudate nucleus and the lowest concentration in the frontal cortex and cerebrospinal fluid. The viral RNA was undetectable in all samples of HIV-negative individuals.

Cutting edge: novel vaccination modality provides significant protection against mucosal infection by highly pathogenic simian immunodeficiency virus.

Vaccine-induced protection against infection by HIV or highly pathogenic and virulent SIV strains has been limited. In a proof-of-concept study, we show that a novel vaccine approach significantly protects rhesus macaques from mucosal infection by the highly pathogenic strain SIVmac251. We vaccinated three cohorts of 12 macaques each with live, irradiated vaccine cells secreting the modified endoplasmic reticulum chaperone gp96-Ig. Cohort 1 was vaccinated with cells secreting gp96SIVIg carrying SIV peptides. In addition, Cohort 2 received recombinant envelope protein SIV-gp120. Cohort 3 was injected with cells secreting gp96-Ig (no SIV Ags) vaccines. Cohort 2 was protected from infection. After seven rectal challenges with highly pathogenic SIVmac251, the hazard ratio was 0.27, corresponding to a highly significant, 73% reduced risk for viral acquisition. The apparent success of the novel vaccine modality recommends further study.

Cell-secreted Gp96-Ig-peptide complexes induce lamina propria and intraepithelial CD8 + cytotoxic T lymphocytes in the intestinal mucosa

Induction of mucosal immunity is critical for protection from enteric pathogens. Heat shock protein gp96 is one of the primary peptide and protein chaperones located in the endoplasmic reticulum. We reported previously that a cell-secreted gp96-Ig fusion protein (gp96-Ig) mediated strong systemic, antigen-specific CD8-CTL expansion in vivo. We now evaluate the mucosal immune response to stimulation by secreted gp96 using allogeneic NIH-3T3 transfected with ovalbumin (OVA) and gp96-Ig. A single intraperitoneal NIH-3T3-OVA-gp96-Ig immunization caused significant homing of OVA-specific TCR transgenic CD8 cells (OT-I) to Peyers patches, to the intraepithelial compartment and to the lamina propria. Intraperitoneal immunization with cells secreting gp96-Ig provided stronger mucosal immunity than the same dose instilled vaginally or rectally or injected subcutaneously or intradermally. Our results provide the first evidence that cell-based gp96-Ig-secreting vaccines may serve as a potent modality to induce mucosal immunity.

Gp96SIVIg immunization induces potent polyepitope specific, multifunctional memory responses in rectal and vaginal mucosa

The ER-resident chaperone gp96, when released by cell lysis, induces an immunogenic chemokine signature and causes innate immune activation of DC and NK cells. Here we show that intraperitoneal immunization with a genetically engineered, secreted form of gp96, gp96-Ig chaperoning SIV antigens, induces high levels of antigen specific CD8 CTL in the rectal and vaginal mucosa of Rhesus macaques. The frequency of SIV Gag- and SIV Tat-tetramer positive CD8 CTL in the intestinal mucosa reached 30-50% after the third immunization. Tetramer positive CD8 CTL expressed appropriate functional (granzyme B) and migration markers (CD103). The polyepitope specificity of the mucosal CD8 and CD4 response is evident from a strong, multifunctional cytokine response upon stimulation with peptides covering the gag, tat and env proteins. Induction of powerful mucosal effector CD8 CTL responses by cell-based gp96(SIV)-Ig immunization may provide a pathway to the development of safe and effective SIV/HIV vaccines.

T Cell Costimulation by TNFR Superfamily (TNFRSF)4 and TNFRSF25 in the Context of Vaccination

TNFR superfamily (TNFRSF)4 (OX40, CD134) and TNFRSF25 are costimulatory receptors that influence CD4+ and CD8+ T cell responses to cognate Ag. Independently, these receptors have been described to stimulate overlapping functions, including enhanced proliferation and activation for both regulatory T cells (CD4+Foxp3+; Tregs) and conventional T cells (CD4+Foxp3− or CD8+Foxp3−; Tconvs). To determine the relative functionality of TNFRSF4 and TNFRSF25 in T cell immunity, the activity of TNFRSF4 and TNFRS25 agonistic Abs was compared in the context of both traditional protein/adjuvant (OVA/aluminum hydroxide) and CD8+-specific heat shock protein-based (gp96-Ig) vaccine approaches. These studies demonstrate that both TNFRSF4 and TNFRSF25 independently and additively costimulate vaccine-induced CD8+ T cell proliferation following both primary and secondary Ag challenge. In contrast, the activities of TNFRSF4 and TNFRSF25 were observed to be divergent in the costimulation of CD4+ T cell immunity. TNFRSF4 agonists were potent costimulators of OVA/aluminum hydroxide-induced CD4+ Tconv proliferation, but they only weakly costimulated Treg proliferation and IgG2a production, whereas TNFRSF25 agonists were strong costimulators of Treg proliferation, producers of IgG1, IgG2a, and IgG2b, and weak costimulators of CD4+ Tconv proliferation. Interestingly, Ag-specific cellular and humoral responses were uncoupled upon secondary immunization, which was dramatically affected by the presence of TNFRSF4 or TNFRSF25 costimulation. These studies highlight the overlapping but nonredundant activities of TNFRSF4 and TNFRSF25 in T cell immunity, which may guide the application of receptor agonistic agents as vaccine adjuvants for infectious disease and tumor immunity.

Tumor Necrosis Factor-alpha Levels in HIV-1 Seropositive Injecting Drug Users

TNF-α is a highly pleiotropic cytokine and plays an important role in regulating HIV-1 replication. It may compromise the integrity of the blood-brain-barrier and, thus, may contribute to the neurotoxicity of HIV-1-infection. Both intravenous drug abuse (IDU) and HIV infection can increase TNF-α activity, but little information is available on the effects of a combination of these factors on TNF-α. We investigated plasma TNF-α levels and mRNA in the peripheral monocytes of 166 men and women in three groups: HIV-1-positive IDUs, HIV-1-negative IDUs, and HIV-negative non-IDU control participants. HIV-1-positive IDUs had higher TNF-α levels than HIV-1-negative IDUs who, in turn, had higher levels than controls. TNF-α mRNA expression in peripheral monocytes was significantly increased in both HIV-1-positive and negative IDUs compared to controls. These findings show that the effects of HIV infection and intravenous drug use may be additive in increasing TNF-α levels. Given the multiple effects of TNF-α in HIV infection, additional investigation of its role is needed.

Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria

Perforin-2 (MPEG1) is a pore-forming, antibacterial protein with broad-spectrum activity. Perforin-2 is expressed constitutively in phagocytes and inducibly in parenchymal, tissue-forming cells. In vitro, Perforin-2 prevents the intracellular replication and proliferation of bacterial pathogens in these cells. Perforin-2 knockout mice are unable to control the systemic dissemination of methicillin-resistant Staphylococcus aureus (MRSA) or Salmonella typhimurium and perish shortly after epicutaneous or orogastric infection respectively. In contrast, Perforin-2-sufficient littermates clear the infection. Perforin-2 is a transmembrane protein of cytosolic vesicles -derived from multiple organelles- that translocate to and fuse with bacterium containing vesicles. Subsequently, Perforin-2 polymerizes and forms large clusters of 100 Å pores in the bacterial surface with Perforin-2 cleavage products present in bacteria. Perforin-2 is also required for the bactericidal activity of reactive oxygen and nitrogen species and hydrolytic enzymes. Perforin-2 constitutes a novel and apparently essential bactericidal effector molecule of the innate immune system. DOI: http://dx.doi.org/10.7554/eLife.06508.001

Humanized mice: novel model for studying mechanisms of human immune-based therapies

The lack of relevant animal models is the major bottleneck for understanding human immunology and immunopathology. In the last few years, a novel model of humanized mouse has been successfully employed to investigate some of the most critical questions in human immunology. We have set up and tested in our laboratory the latest technology for generating mice with a human immune system by reconstituting newborn immunodeficient NOD/SCID-γc−/− mice with human fetal liver-derived hematopoietic stem cells. These humanized mice have been deemed most competent as human models in a thorough comparative study with other humanized mouse technologies. Lymphocytes in these mice are of human origin while other hematopoietic cells are chimeric, partly of mouse and partly of human origin. We demonstrate that human CD8 T lymphocytes in humanized mice are fully responsive to our novel cell-based secreted heat shock protein gp96HIV-Ig vaccine. We also show that the gp96HIV-Ig vaccine induces powerful mucosal immune responses in the rectum and the vagina, which are thought to be required for protection from HIV infection. We posit the hypothesis that vaccine approaches tested in humanized mouse models can generate data rapidly, economically and with great flexibility (genetic manipulations are possible), to be subsequently tested in larger nonhuman primate models and humans.

Induction of IL21 in Peripheral T Follicular Helper Cells Is an Indicator of Influenza Vaccine Response in a Previously Vaccinated HIV-Infected Pediatric Cohort

HIV-infected patients of all ages frequently underperform in response to seasonal influenza vaccination, despite virologic control of HIV. The molecular mechanisms governing this impairment, as well as predictive biomarkers for responsiveness, remain unknown. This study was performed in samples obtained prevaccination (T0) from HIV-infected children who received the 2012–2013 seasonal influenza vaccine. Response status was determined based on established criterion for hemagglutination inhibition titer; participants with a hemagglutination titer ≥1:40 plus a ≥4-fold increase over T0 at 3 wk postvaccination were designated as responders. All children had a history of prior influenza vaccinations. At T0, the frequencies of CD4 T cell subsets, including peripheral T follicular helper (pTfh) cells, which provide help to B cells for developing into Ab-secreting cells, were similar between responders and nonresponders. However, in response to in vitro stimulation with influenza A/California/7/2009 (H1N1) Ag, differential gene expression related to pTfh cell function was observed by Fluidigm high-density RT-PCR between responders and nonresponders. In responders, H1N1 stimulation at T0 also resulted in CXCR5 induction (mRNA and protein) in CD4 T cells and IL21 gene induction in pTfh cells that were strongly associated with H1N1-specific B cell responses postvaccination. In contrast, CD4 T cells of nonresponders exhibited increased expression of IL2 and STAT5 genes, which are known to antagonize peripheral Tfh cell function. These results suggest that the quality of pTfh cells at the time of immunization is important for influenza vaccine responses and provide a rationale for targeted, ex vivo Ag-driven molecular profiling of purified immune cells to detect predictive biomarkers of the vaccine response.