During chronic HIV-1 infection, the upregulation of immune checkpoints (ICs) on lymphocytes contributes to persistent immune dysfunction, T-cell exhaustion, and inadequate virological control, highlighting the potential role of ICs in HIV functional cure strategy. Despite extensive research on ICs, the expression dynamics and immunological impact of ICs in natural HIV/SHIV infection, particularly among antiretroviral therapy (ART)-free elite controllers (ECs), remain poorly understood. In this study, we monitored a cohort of SHIVSF162P3-infected rhesus macaques (RMs), identifying four ECs and four progressors (PGs) through longitudinal evaluation. We observed low-level expression of ICs in both peripheral blood and lymph nodes of ECs, characterized by a particularly pronounced restriction in the expression of TIGIT and BTLA. This attenuated IC profile correlated with enhanced T-cell functionality, reduced exhaustion markers, and reduced viral reservoirs in peripheral and lymphoid tissues. Transcriptomic profiling revealed that TIGIT is a critical checkpoint marker involved in multiple synergistic cofunctions related to HIV/SIV-specific immune regulation. Collectively, our findings establish a dual role for ICs in perpetuating T-cell exhaustion and viral reservoir persistence, paving the way for IC blockade in future HIV cure strategies.IMPORTANCERhesus macaques spontaneously controlling simian-human immunodeficiency virus (SHIV) without antiretroviral therapy have low-level expression of immune molecules (ICs), characterized by TIGIT and BTLA. These molecules are linked to enhanced immune function and reduced viral presence in peripheral blood and lymph nodes. Transcriptomic profiling revealed that TIGIT is a critical checkpoint marker involved in multiple synergistic cofunctions related to HIV/SIV-specific immune regulation in both humans and macaques. Blocking TIGIT improved polyfunctional T-cell responses, thereby offering a potential new treatment strategy and providing critical insights for developing a functional HIV cure.

Transmembrane 4 L six family member 1 (TM4SF1) is highly expressed and contributes to the progression of various malignancies. However, how it modulates hepatocellular carcinoma (HCC) progression and senescence remains to be elucidated.
TM4SF1 expression in HCC samples was evaluated using immunohistochemistry and flow cytometry. Cellular senescence was assessed through SA-β-gal activity assays and Western blot analysis. TM4SF1-related protein interactions were investigated using immunoprecipitation-mass spectrometry, co-immunoprecipitation, bimolecular fluorescence complementation, and immunofluorescence. Tumor-infiltrating immune cells were analyzed by flow cytometry. The HCC mouse model was established via hydrodynamic tail vein injection.
TM4SF1 was highly expressed in human HCC samples and murine models. Knockdown of TM4SF1 suppressed HCC proliferation both in vitro and in vivo, inducing non-secretory senescence through upregulation of p16 and p21. TM4SF1 enhanced the interaction between AKT1 and PDPK1, thereby promoting AKT phosphorylation, which subsequently downregulated p16 and p21. Meanwhile, TM4SF1-mediated AKT phosphorylation enhanced PD-L1 expression while reducing major histocompatibility complex class I level on tumor cells, leading to impaired cytotoxic function of CD8+ T cells and an increased proportion of exhausted CD8+ T cells. In clinical HCC samples, elevated TM4SF1 expression was associated with resistance to anti-PD-1 immunotherapy. Targeting TM4SF1 via adeno-associated virus induced tumor senescence, reduced tumor burden and synergistically enhanced the efficacy of anti-PD-1 therapy.
Our results revealed that TM4SF1 regulated tumor cell senescence and immune evasion through the AKT pathway, highlighting its potential as a therapeutic target in HCC, particularly in combination with first-line immunotherapy.

Strong cellular immunity contributes to the control of HIV infection. Here, we describe a step-by-step protocol to assess the simian immunodeficiency virus (SIV)-specific CD8+ T cell responses by quantifying the degranulation, cytokine and chemokine production from SHIVSF162P3-infected rhesus macaques with an HIV fusion-inhibitory lipopeptide (LP-98) monotherapy. We also present the steps for adoptive transfer of an anti-CD8 antibody into a stable virologic control (SVC) group of LP-98-treated monkeys, confirming a direct role of CD8+ T cells in SVC macaques. For complete details on the use and execution of this protocol, please refer to Xue et al. (2022).
Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.

Accumulation of activated natural killer (NK) cells in tissues during Ebola virus infection contributes to Ebola virus disease (EVD) pathogenesis. Yet, immunization with Ebola virus-like particles (VLPs) comprising glycoprotein and matrix protein VP40 provides rapid, NK cell-mediated protection against Ebola challenge. We used Ebola VLPs as the viral surrogates to elucidate the molecular mechanism by which Ebola virus triggers heightened NK cell activity. Incubation of human peripheral blood mononuclear cells with Ebola VLPs or VP40 protein led to increased expression of IFN-γ, TNF-α, granzyme B, and perforin by CD3-CD56+ NK cells, along with increases in degranulation and cytotoxic activity of these cells. Optimal activation required accessory cells like CD14+ myeloid and CD14- cells and triggered increased secretion of numerous inflammatory cytokines. VP40-induced IFN-γ and TNF-α secretion by NK cells was dependent on IL-12 and IL-18 and suppressed by IL-10. In contrast, their increased degranulation was dependent on IL-12 with little influence of IL-18 or IL-10. These results demonstrate that Ebola VP40 stimulates NK cell functions in an IL-12- and IL-18-dependent manner that involves CD14+ and CD14- accessory cells. These potentially novel findings may help in designing improved intervention strategies required to control viral transmission during Ebola outbreaks.

Interleukin-2 (IL-2) is critical for regulatory T cell (Treg) function and homeostasis. At low doses, IL-2 can suppress immune pathologies by expanding Tregs that constitutively express the high affinity IL-2Rα subunit. However, even low dose IL-2, signaling through the IL2-Rβ/γ complex, may lead to the activation of proinflammatory, non-Treg T cells, so improving specificity toward Tregs may be desirable. Here we use messenger RNAs (mRNA) to encode a half-life-extended human IL-2 mutein (HSA-IL2m) with mutations promoting reliance on IL-2Rα. Our data show that IL-2 mutein subcutaneous delivery as lipid-encapsulated mRNA nanoparticles selectively activates and expands Tregs in mice and non-human primates, and also reduces disease severity in mouse models of acute graft versus host disease and experimental autoimmune encephalomyelitis. Single cell RNA-sequencing of mouse splenic CD4+ T cells identifies multiple Treg states with distinct response dynamics following IL-2 mutein treatment. Our results thus demonstrate the potential of mRNA-encoded HSA-IL2m immunotherapy to treat autoimmune diseases.
© 2022. The Author(s).