Here, we present a protocol to evaluate the killing capacity and functional profile of human HIV-specific CD8 T cells. We describe steps for culturing peripheral blood mononuclear cells (PBMCs) from patients with HIV on antiretroviral therapy (ART) with HIV peptides ex vivo and quantifying HIV-specific CD8 T cell killing using flow cytometry. We then detail procedures for integrating the established killing assay with intracellular cytokine staining (ICS) and assessing CD8 T cell function. This protocol can provide insights into CD8 T cell-mediated immunity against HIV. For complete details on the use and execution of this protocol, please refer to Mbitikon-Kobo et al.,1 Noto et al.,2 and Gubser et al.3.
Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.

Oncolytic virus (OV) clinical trials have demonstrated remarkable efficacy in subsets of patients with glioblastoma (GBM). However, the lack of tools to predict this response hinders the advancement of a more personalized application of OV therapy. In this study, we characterize an ex vivo co-culture system designed to examine the immune response to OV infection of patient-derived GBM neurospheres in the presence of autologous peripheral blood mononuclear cells (PBMCs). Co-culture conditions were optimized to retain viability and functionality of both tumor cells and PBMCs, effectively recapitulating the well-recognized immunosuppressive effects of GBM. Following OV infection, we observed elevated secretion of pro-inflammatory cytokines and chemokines, including interferon γ, tumor necrosis factor α, CXCL9, and CXCL10, and marked changes in immune cell activation markers. Importantly, OV treatment induced unique patient-specific immune responses. In summary, our co-culture platform presents an avenue for personalized screening of viro-immunotherapies in GBM, offering promise as a potential tool for future patient stratification in OV therapy.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Glucocorticoid-induced tumor necrosis factor related protein (GITR) is a co-stimulatory immune checkpoint molecule constitutively expressed on regulatory T cells (Tregs) and on activated T conventional cells (Tconv). In blood collected from PWH on suppressive ART, GITR expression was reduced in multiple activated CD4 and CD8 T cell subsets but was increased in Tregs. HIV specific CD8 T cells expressed higher levels of GITR and programmed cell death protein 1 (PD-1) compared to total CD8 T cells. Following stimulation with HIV peptides and GITR-ligand (L), we demonstrated a significant decrease in killing by HIV specific CD8 T cells and an increased exhausted profile. T cell receptor co-stimulation with GITR-L abrogated Treg suppression and induced expansion of CD4 Tconv. We conclude that GITR activation is an additional factor contributing to an impaired HIV immune response in PWH on ART and that GITR agonist antibodies should not be pursued for HIV cure strategies.
© 2023 The Authors.

Preclinical studies demonstrate that pharmacological mobilization and recruitment of endogenous bone marrow stem cells and immunoregulatory cells by a fixed-dose drug combination (MRG-001) improves wound healing, promotes tissue regeneration, and prevents allograft rejection. In this phase I, first-in-human study, three cohorts receive subcutaneous MRG-001 or placebo, every other day for 5 days. The primary outcome is safety and tolerability of MRG-001. Fourteen subjects received MRG-001 and seven received a placebo. MRG-001 is safe over the selected dose range. There are no clinically significant laboratory changes. The intermediate dose group demonstrates the most significant white blood cell, stem cell, and immunoregulatory cell mobilization. PBMC RNA sequencing and gene set enrichment analysis reveal 31 down-regulated pathways in the intermediate MRG-001 dose group compared with no changes in the placebo group. MRG-001 is safe across all dose ranges. MRG-001 may be a clinically useful therapy for immunoregulation and tissue regeneration (ClinicalTrials.gov: NCT04646603).
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

A complete chart of the chromatin regulatory elements of immune cells in patients with cancer and their dynamic behavior is necessary to understand the developmental fates and guide therapeutic strategies. Here, we map the single-cell chromatin landscape of immune cells from blood, normal tumor-adjacent kidney tissue and malignant tissue from patients with early-stage clear cell renal cell carcinoma (ccRCC). We catalog the T cell states dictated by tissue-specific and developmental-stage-specific chromatin accessibility patterns, infer key chromatin regulators and observe rewiring of regulatory networks in the progression to dysfunction in CD8+ T cells. Unexpectedly, among the transcription factors orchestrating the path to dysfunction, NF-κB is associated with a pro-apoptotic program in late stages of dysfunction in tumor-infiltrating CD8+ T cells. Importantly, this epigenomic profiling stratified ccRCC patients based on a NF-κB-driven pro-apoptotic signature. This study provides a rich resource for understanding the functional states and regulatory dynamics of immune cells in ccRCC.
© 2022. The Author(s).