T cells mediate cell non-autonomous arterial ageing in mice.

KEY POINTS: Increased large artery stiffness and impaired endothelium-dependent dilatation occur with advanced age. We sought to determine whether T cells mechanistically contribute to age-related arterial dysfunction. We found that old mice exhibited greater proinflammatory T cell accumulation around both the aorta and mesenteric arteries. Pharmacologic depletion or genetic deletion of T cells in old mice resulted in ameliorated large artery stiffness and greater endothelium-dependent dilatation compared with mice with T cells intact. ABSTRACT: Ageing of the arteries is characterized by increased large artery stiffness and impaired endothelium-dependent dilatation. T cells contribute to hypertension in acute rodent models but whether they contribute to chronic age-related arterial dysfunction is unknown. To determine whether T cells directly mediate age-related arterial dysfunction, we examined large elastic artery and resistance artery function in young (4-6 months) and old (22-24 months) wild-type mice treated with anti-CD3 F(ab'2) fragments to deplete T cells (150 µg, i.p. every 7 days for 28 days) or isotype control fragments. Old mice exhibited greater numbers of T cells in both aorta and mesenteric vasculature when compared with young mice. Old mice treated with anti-CD3 fragments exhibited depletion of T cells in blood, spleen, aorta and mesenteric vasculature. Old mice also exhibited greater numbers of aortic and mesenteric IFN-γ and TNF-α-producing T cells when compared with young mice. Old control mice exhibited greater large artery stiffness and impaired resistance artery endothelium-dependent dilatation in comparison with young mice. In old mice, large artery stiffness was ameliorated with anti-CD3 treatment. Anti-CD3-treated old mice also exhibited greater endothelium-dependent dilatation than age-matched controls. We also examined arterial function in young and old Rag-1-/- mice, which lack lymphocytes. Rag-1-/- mice exhibited blunted increases in large artery stiffness with age compared with wild-type mice. Old Rag-1-/- mice also exhibited greater endothelium-dependent dilatation compared with old wild-type mice. Collectively, these results demonstrate that T cells play an important role in age-related arterial dysfunction.

T lymphocyte depletion ameliorates age-related metabolic impairments in mice.

Both glucose tolerance and adaptive immune function exhibit significant age-related alterations. The influence of the immune system on obesity-associated glucose intolerance is well characterized; however, whether the immune system contributes to age-related glucose intolerance is not as well understood. Here, we report that advancing age results in an increase in T cell infiltration in the epididymal white adipose tissue (eWAT), liver, and skeletal muscle. Subtype analyses show that both CD4+, CD8+ T cells are greater with advancing age in each of these tissues and that aging results in a blunted CD4 to CD8 ratio. Anti-CD3 F(ab')2 fragments depleted CD4+ and CD8+ cells in eWAT, CD4+ cells only in the liver, and did not deplete quadriceps T cells. In old mice, T cells producing both interferon-γ and tumor necrosis factor-α are accumulated in the eWAT and liver, and a greater proportion of skeletal muscle T cells produced interferon-γ. Aging resulted in increased proportion and numbers of T regulatory cells in eWAT, but not in the liver or muscle. Aging also resulted in greater numbers of eWAT and quadriceps CD206- macrophages and eWAT, liver and quadriceps B cells; neither cell type was altered by anti-CD3 treatment. Anti-CD3 treatment improved glucose tolerance in old mice and was accompanied by improved signaling related to liver and skeletal muscle insulin utilization and decreased gluconeogenesis-related gene expression in the liver. Our findings indicate a critical role of the adaptive immune system in the age-related metabolic dysfunction.

Structure-Activity Relationship Analysis of Benzotriazine Analogues as HIV-1 Latency-Reversing Agents.

"Shock and kill" therapeutic strategies toward HIV eradication are based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) and the consequent killing of the reactivated cell by either the cytopathic effect of HIV or an arm of the immune system. We have recently found several benzotriazole and benzotriazine analogues that have the ability to reactivate latent HIV by inhibiting signal transducer and activator of transcription 5 (STAT5) SUMOylation and promoting STAT5 binding to the HIV long terminal repeat and increasing its transcriptional activity. To understand the essential structural groups required for biological activity of these molecules, we performed a systematic analysis of >40 analogues. First, we characterized the essential motifs within these molecules that are required for their biological activity. Second, we identified three benzotriazine analogues with similar activity. We demonstrated that these three compounds are able to increase STAT5 phosphorylation and transcriptional activity. All active analogues reactivate latent HIV in a primary cell model of latency and enhance the ability of interleukin-15 to reactivate latent HIV in cells isolated from aviremic participants. Third, this family of compounds also promote immune effector functions in vitro in the absence of toxicity or global immune activation. Finally, initial studies in mice suggest lack of acute toxicity in vivo A better understanding of the biological activity of these compounds will help in the design of improved LRAs that work via inhibition of STAT5 SUMOylation.

Dual TLR2 and TLR7 agonists as HIV latency-reversing agents.

The presence of a reservoir of latently infected cells in HIV-infected patients is a major barrier towards finding a cure. One active cure strategy is to find latency-reversing agents that induce viral reactivation, thus leading to immune cell recognition and elimination of latently infected cells, known as the shock-and-kill strategy. Therefore, the identification of molecules that reactivate latent HIV and increase immune activation has the potential to further these strategies into the clinic. Here, we characterized synthetic molecules composed of a TLR2 and a TLR7 agonist (dual TLR2/7 agonists) as latency-reversing agents and compared their activity with that of the TLR2 agonist Pam2CSK4 and the TLR7 agonist GS-9620. We found that these dual TLR2/7 agonists reactivate latency by 2 complementary mechanisms. The TLR2 component reactivates HIV by inducing NF-κB activation in memory CD4+ T cells, while the TLR7 component induces the secretion of TNF-α by monocytes and plasmacytoid dendritic cells, promoting viral reactivation in CD4+ T cells. Furthermore, the TLR2 component induces the secretion of IL-22, which promotes an antiviral state and blocks HIV infection in CD4+ T cells. Our study provides insight into the use of these agonists as a multipronged approach targeting eradication of latent HIV.

Influence of Biological Sex, Age, and HIV Status in an In Vitro Primary Cell Model of HIV Latency Using a CXCR4 Tropic Virus.

Primary cell models of human immunodeficiency virus (HIV) latency have become tools to both understand the mechanisms involved in establishment of latency and test preclinical strategies toward HIV-1 cure. These models rely on infection of CD4 T cells from healthy donors. As such, these models provide an opportunity to explore the role of biological sex, age, and HIV status on establishment and reactivation of latent HIV in vitro. We have used an established primary cell model of latency based on the generation of latently infected central memory CD4 T cells with the CXCR4 strain HIV-1NL4-3 to address whether these variables influence (i) HIV-1NL4-3 replication, (ii) establishment of latency, and (iii) latency reversal in CD4 T cells. Our results indicate that replication of HIV-1NL4-3, but not establishment of latency, is influenced by the age of female, but not male, donors. Moreover, the frequency of latently infected cells in this model is directly correlated with levels of productive infection in both male and female donors independent of age. We did not find differences in the ability of five different latency-reversing agents to reactivate latent HIV-1NL4-3. Finally, we have found that this model can be generated using cells from aviremic participants. In conclusion, we have further characterized the central memory T cell model of latency regarding biological sex and age and demonstrated that this model is suitable for use with cells isolated from aviremic participants, opening the opportunity to use this primary cell model to address cure approaches, including shock and kill, in HIV-infected individuals.