Immune restoration by TIGIT blockade is insufficient to control chronic SIV infection.

TIGIT is a negative immune checkpoint receptor associated with T cell exhaustion in cancer and HIV. TIGIT upregulation in virus-specific CD8+ T cells and NK cells during HIV/SIV infection results in dysfunctional effector capabilities. In vitro studies targeting TIGIT on CD8+ T cells suggest TIGIT blockade as a viable strategy to restore SIV-specific T cell responses. Here, we extend these studies in vivo using TIGIT blockage in nonhuman primates in an effort to reverse T cell and NK cell exhaustion in the setting of SIV infection. We demonstrate that in vivo administration of a humanized anti-TIGIT monoclonal antibody (mAb) is well tolerated in both cynomolgus macaques and rhesus macaques. Despite sustained plasma concentrations of anti-TIGIT mAb, we observed no consistent improvement in NK or T cell cytolytic capacity. TIGIT blockade minimally enhanced T cell proliferation and virus-specific T cell responses in both magnitude and breadth though plasma viral loads in treated animals remained stable indicating that anti-TIGIT mAb treatment alone was insufficient to increase anti-SIV CD8+ T cell function. The enhancement of virus-specific T cell proliferative responses observed in vitro with single or dual blockade of TIGIT and/or PD-1 highlights TIGIT as a potential target to reverse T cell dysfunction. Our studies, however, reveal that targeting the TIGIT pathway alone may be insufficient in the setting of viremia and that combining immune checkpoint blockade with other immunotherapeutics may be a future path forward for improved viral control or elimination of HIV.IMPORTANCEUpregulation of the immune checkpoint receptor TIGIT is associated with HIV-mediated T cell dysfunction and correlates with HIV disease progression. Compelling evidence exists for targeting immune checkpoint receptor pathways that would potentially enhance immunity and refocus effector cell efforts toward viral clearance. In this report, we investigate TIGIT blockade as an immunotherapeutic approach to reverse immune exhaustion during chronic SIV/SHIV infection in a nonhuman primate model of HIV infection. We show that interfering with the TIGIT signaling axis alone is insufficient to improve viral control despite modest improvement in T cell immunity. Our data substantiate the use of targeting multiple immune checkpoint receptors to promote synergy and ultimately eliminate HIV-infected cells.

Allogeneic immunity clears latent virus following allogeneic stem cell transplantation in SIV-infected ART-suppressed macaques.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) from donors lacking C-C chemokine receptor 5 (CCR5Δ32/Δ32) can cure HIV, yet mechanisms remain speculative. To define how alloHSCT mediates HIV cure, we performed MHC-matched alloHSCT in SIV+, anti-retroviral therapy (ART)-suppressed Mauritian cynomolgus macaques (MCMs) and demonstrated that allogeneic immunity was the major driver of reservoir clearance, occurring first in peripheral blood, then peripheral lymph nodes, and finally in mesenteric lymph nodes draining the gastrointestinal tract. While allogeneic immunity could extirpate the latent viral reservoir and did so in two alloHSCT-recipient MCMs that remained aviremic >2.5 years after stopping ART, in other cases, it was insufficient without protection of engrafting cells afforded by CCR5-deficiency, as CCR5-tropic virus spread to donor CD4+ T cells despite full ART suppression. These data demonstrate the individual contributions of allogeneic immunity and CCR5 deficiency to HIV cure and support defining targets of alloimmunity for curative strategies independent of HSCT.

Evaluation of Effects of Laboratory Disinfectants on Mouse Gut Microbiota.

Disturbances in the gut microbiota are known to be associated with numerous human diseases. Mice have proven to be an invaluable tool for investigating the role of the gut microbiota in disease processes. Nonexperimental factors related to maintaining mice in the laboratory environment are increasingly being shown to have inadvertent effects on the gut microbiota and may function as confounding variables. Microisolation technique is a term used to describe the common biosecurity practice of spraying gloved hands with disinfectant before handling research mice. This practice prevents contamination with pathogenic microorganisms. To investigate if exposure to disinfectants can affect the mouse gut microbiota, C57BL/6 mice were exposed daily for 27 consecutive days to commonly used laboratory disinfectants through microisolation technique. The effects of 70% ethanol and disinfectant products containing chlorine dioxide, hydrogen peroxide, or potassium peroxymonosulfate were each evaluated. Fecal pellets were collected after 7, 14, 21, and 28 d of disinfectant exposure, and cecal contents were collected at day 28. DNA extractions were performed on all cecal and fecal samples, and microbial community structure was characterized using 16S ribosomal RNA amplicon sequencing. Alpha and ß diversity metrics and taxon-level analyses were used to evaluate differences in microbial communities. Disinfectant had a small but significant effect on fecal microbial communities compared with sham-exposed controls, and effects varied by disinfectant type. In general, longer exposure times resulted in greater changes in the fecal microbiota. Effects on the cecal microbiota were less pronounced and only seen with the hydrogen peroxide and potassium peroxymonosulfate disinfectants. These results indicate that laboratory disinfectant use should be considered as a potential factor that can affect the mouse gut microbiota.

Reference Intervals for Total T4 and Free T4 in Cynomolgus Macaques (Macaca fascicularis) and Rhesus Macaques (Macaca mulatta).

Thyroid diseases, associated with either increased or decreased concentrations of circulating thyroid hormones, are prevalent in both human and veterinary populations. Hypothyroidism is a differential diagnosis for many medical problems as the disease presents with nonspecific clinical signs that can include lethargy, weight gain, cold intolerance, and dermatologic manifestations such as alopecia. Alopecia is a frequently reported problem in captive nonhuman primates (NHP), and hypothyroidism is considered to be a differential diagnosis. However, thyroid function test results in NHP using total T4 (TT4) and free T4 (FT4) assays are difficult to interpret without accurate reference intervals (RI) for comparison. As a consequence, hypothyroidism may be underdiagnosed in these species. The objective of this study was to establish RI for TT4 and FT4 in healthy populations of cynomolgus macaques ( n = 133; age range 2.6 to 24.7 y) and rhesus macaques ( n = 172; age range 0.8 to 31.0 y). Serum samples were collected across a 14-y period during routine anesthetic events in clinically healthy animals, and TT4 and FT4 concentrations were measured using commercially available immunoassays. The RI established for TT4 and FT4 were 5.1 to 14.9 ug/dL and 0.48 to 1.17 ng/dL for cynomolgus macaques, and 3.9 to 14.7 ug/dL and 0.36 to 1.12 ng/dL for rhesus macaques. Significant differences in thyroid hormone concentrations were found between Indian and Chinese origin rhesus, and between Mauritian and other origin cynomolgus. In addition, juvenile and subadult rhesus exhibited significantly higher FT4 and TT4 concentrations than did older animals. Individual RI were established for subgroups with adequately different thyroid hormone concentrations. These results will allow a more thorough diagnostic evaluation of cynomolgus and rhesus macaques with clinical signs consistent with thyroid disease and will ultimately be a refinement in NHP medicine.

TLR5 participates in the TLR4 receptor complex and promotes MyD88-dependent signaling in environmental lung injury.

Lung disease causes significant morbidity and mortality, and is exacerbated by environmental injury, for example through lipopolysaccharide (LPS) or ozone (O3). Toll-like receptors (TLRs) orchestrate immune responses to injury by recognizing pathogen- or danger-associated molecular patterns. TLR4, the prototypic receptor for LPS, also mediates inflammation after O3, triggered by endogenous hyaluronan. Regulation of TLR4 signaling is incompletely understood. TLR5, the flagellin receptor, is expressed in alveolar macrophages, and regulates immune responses to environmental injury. Using in vivo animal models of TLR4-mediated inflammations (LPS, O3, hyaluronan), we show that TLR5 impacts the in vivo response to LPS, hyaluronan and O3. We demonstrate that immune cells of human carriers of a dominant negative TLR5 allele have decreased inflammatory response to O3 exposure ex vivo and LPS exposure in vitro. Using primary murine macrophages, we find that TLR5 physically associates with TLR4 and biases TLR4 signaling towards the MyD88 pathway. Our results suggest an updated paradigm for TLR4/TLR5 signaling.

Immune cells in the lung help guard against infections. On the surface of these cells are proteins called TLR receptors that recognize dangerous molecules or DNA from disease-causing microbes such as bacteria. When the immune cells detect these invaders, the TLR receptors spring into action and trigger an inflammatory response to destroy the microbes. This inflammation usually helps the lung clear infections. But it can also be harmful and damage the lung, for example when inflammation is caused by non-infectious substances such as pollutants in the atmosphere. There are several TLR receptors that each recognize a specific molecule. In 2010, researchers showed that the receptor TLR4 is responsible for causing inflammation in the lung after exposure to pollution. Another receptor called TLR5 also helps activate the immune response in the lung. But it was unclear whether this receptor also plays a role in pollution-linked lung damage. Now, Hussain, Johnson, Sciurba et al. ­ including one of the researchers involved in the 2010 study ­ have investigated the role of TLR5 in immune cells from the lungs of humans and mice. The experiments showed that TLR5 works together with TLR4 and helps trigger an inflammatory response to both pollutants and bacteria. Hussain et al. found that people lacking a working TLR5 receptor (which make up 3­10% of the population) are less likely to experience lung inflammation when exposed to pollution or bacterial proteins that activate TLR4. These findings suggest that people without TLR5 may be protected from pollution-induced lung injury. Further research into the role of TLR5 could help develop genetic tests for identifying people who are more sensitive to damage from pollution. This information could then be used to determine the likelihood of a patient experiencing certain lung diseases.

Activation of human mesenchymal stem cells impacts their therapeutic abilities in lung injury by increasing interleukin (IL)-10 and IL-1RN levels.

Acute respiratory distress syndrome (ARDS) is an important cause of morbidity and mortality, with no currently effective therapies. Several preclinical studies have shown that human mesenchymal stem cells (hMSCs) have therapeutic potential for patients with ARDS because of their immunomodulatory properties. The clinical use of hMSCs has some limitations, such as the extensive manipulation required to isolate the cells from bone marrow aspirates and the heterogeneity in their anti-inflammatory effect in animal models and clinical trials. The objective of this study was to improve the protective anti-inflammatory capacity of hMSCs by evaluating the consequences of preactivating hMSCs before use in a murine model of ARDS. We injected endotoxemic mice with minimally manipulated hMSCs isolated from the bone marrow of vertebral bodies with or without prior activation with serum from ARDS patients. Minimally manipulated hMSCs were more efficient at reducing lung inflammation compared with isolated and in vitro expanded hMSCs obtained from bone marrow aspirates. Where the most important effect was observed was with the activated hMSCs, independent of their source, which resulted in increased expression of interleukin (IL)-10 and IL-1 receptor antagonist (RN), which was associated with enhancement of their protective capacity by reduction of the lung injury score, development of pulmonary edema, and accumulation of bronchoalveolar lavage inflammatory cells and cytokines compared with nonactivated cells. This study demonstrates that a low manipulation during hMSC isolation and expansion increases, together with preactivation prior to the therapeutic use of hMSCs, would ensure an appropriate immunomodulatory phenotype of the hMSCs, reducing the heterogeneity in their anti-inflammatory effect.