Cellular redox state determinants are traditionally studied using fluorescent microscopy and immunoblot analysis; however, no procedure has been developed for simultaneous measurement in various immune cell subsets. Here, we present a flow cytometry assay for measuring antioxidant defense systems and reactive oxygen species simultaneously in T, B, and natural killer lymphocytes. We describe steps for preparing and treating peripheral blood mononuclear cells, surface and dye staining, cell fixation/permeabilization, and intracellular staining. We then detail machine standardization, acquisition, and analysis.
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

Intercellular adhesion molecule 1 (ICAM-1) plays prominent roles in mediating cell-cell adhesion which also facilitates B cell activation and differentiation with the help from CD4+ T cells. Here, we have reported a unique phenomenon that increased ICAM-1 on purified human CD4+ T cells upon anti-CD3/CD28 stimulation enhanced CD4+ T-B cell adhesion whereas induced less B cell differentiation and IgG production. This was largely due to increased PD-1 expression on CD19hi B cells after coculturing with hyperactivated CD4+ T cells. Consequently, ICAM-1 blockade during CD4+ T cell-B cell coculture promoted IgG production with the activation of ERK1/2 and Blimp-1/IRF4 upregulation. Consistently, CD4+ T cells from moderate-to-severe SLE patients with high ICAM-1 expression mediated less IgG production after T-B coculture. Therefore, ICAM-1-mediated human CD4+ T-B cell adhesion provides dual roles on B cell differentiation and IgG production partially depending on expression levels of PD-1 on B cells, supporting cell adhesion and subsequent PD-1 induction as an alternative intrinsic checkpoint for B cell differentiation.
© 2023 The Authors.

Spontaneous transcription and translation of HIV can persist during suppressive antiretroviral therapy (ART). The quantity, phenotype, and biological relevance of this spontaneously "active" reservoir remain unclear. Using multiplexed single-cell RNAflow-fluorescence in situ hybridization (FISH), we detect active HIV transcription in 14/18 people with HIV on suppressive ART, with a median of 28/million CD4+ T cells. While these cells predominantly exhibit abortive transcription, p24-expressing cells are evident in 39% of participants. Phenotypically diverse, active reservoirs are enriched in central memory T cells and CCR6- and activation-marker-expressing cells. The magnitude of the active reservoir positively correlates with total HIV-specific CD4+ and CD8+ T cell responses and with multiple HIV-specific T cell clusters identified by unsupervised analysis. These associations are particularly strong with p24-expressing active reservoir cells. Single-cell vDNA sequencing shows that active reservoirs are largely dominated by defective proviruses. Our data suggest that these reservoirs maintain HIV-specific CD4+ and CD8+ T responses during suppressive ART.
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

An efficacious HIV vaccine will need to elicit a complex package of innate, humoral, and cellular immune responses. This complex package of responses to vaccine candidates has been studied and yielded important results, yet it has been a recurring challenge to determine the magnitude and protective effect of specific in vivo immune responses in isolation. We therefore designed a single, viral-spike-apical, epitope-focused V2 loop immunogen to reveal individual vaccine-elicited immune factors that contribute to protection against HIV/SIV.
We generated a novel vaccine by incorporating the V2 loop B-cell epitope in the cholera toxin B (CTB) scaffold and compared two new immunization regimens to a historically protective 'standard' vaccine regimen (SVR) consisting of 2xDNA prime boosted with 2xALVAC-SIV and 1xΔV1gp120. We immunized a cohort of macaques with 5xCTB-V2c vaccine+alum intramuscularly simultaneously with topical intrarectal vaccination of CTB-V2c vaccine without alum (5xCTB-V2/alum). In a second group, we tested a modified version of the SVR consisting of 2xDNA prime and boosted with 1xALVAC-SIV and 2xALVAC-SIV+CTB-V2/alum, (DA/CTB-V2c/alum).
In the absence of any other anti-viral antibodies, V2c epitope was highly immunogenic when incorporated in the CTB scaffold and generated highly functional anti-V2c antibodies in the vaccinated animals. 5xCTB-V2c/alum vaccination mediated non-neutralizing ADCC activity and efferocytosis, but produced low avidity, trogocytosis, and no neutralization of tier 1 virus. Furthermore, DA/CTB-V2c/alum vaccination also generated lower total ADCC activity, avidity, and neutralization compared to the SVR. These data suggest that the ΔV1gp120 boost in the SVR yielded more favorable immune responses than its CTB-V2c counterpart. Vaccination with the SVR generates CCR5- α4β7+CD4+ Th1, Th2, and Th17 cells, which are less likely to be infected by SIV/HIV and likely contributed to the protection afforded in this regimen. The 5xCTB-V2c/alum regimen likewise elicited higher circulating CCR5- α4β7+ CD4+ T cells and mucosal α4β7+ CD4+ T cells compared to the DA/CTB-V2c/alum regimen, whereas the first cell type was associated with reduced risk of viral acquisition.
Taken together, these data suggest that individual viral spike B-cell epitopes can be highly immunogenic and functional as isolated immunogens, although they might not be sufficient on their own to provide full protection against HIV/SIV infection.
Copyright © 2023 Rahman, Becerra-Flores, Patskovsky, Silva de Castro, Bissa, Basu, Shen, Williams, Sarkis, N’guessan, LaBranche, Tomaras, Aye, Veazey, Paquin-Proulx, Rao, Franchini and Cardozo.

The human immunodeficiency virus epidemic continues in sub-Saharan Africa, and particularly affects adolescent girls and women who have limited access to antiretroviral therapy. Here we report that the risk of vaginal simian immunodeficiency virus (SIV)mac251 acquisition is reduced by more than 90% using a combination of a vaccine comprising V1-deleted (V2 enhanced) SIV envelope immunogens with topical treatment of the zinc-finger inhibitor SAMT-247. Following 14 weekly intravaginal exposures to the highly pathogenic SIVmac251, 80% of a cohort of 20 macaques vaccinated and treated with SAMT-247 remained uninfected. In an arm of 18 vaccinated-only animals without microbicide, 40% of macaques remained uninfected. The combined SAMT-247/vaccine regimen was significantly more effective than vaccination alone. By analysing immune correlates of protection, we show that, by increasing zinc availability, SAMT-247 increases natural killer cytotoxicity and monocyte efferocytosis, and decreases T-cell activation to augment vaccine-induced protection.
© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.