Young females are at high risk of acquiring HIV-1 infections and an imbalance in the vaginal microbiome enhances susceptibility to HIV-1 infection. More insights into the underlying mechanisms could open up new strategies to prevent HIV-1 acquisition and dissemination. Here, we investigated the effect of anaerobic bacteria associated with bacterial vaginosis (BV) on HIV-1 transmission by two distinct dendritic cell (DC) subsets, that is, inflammatory monocyte-derived DCs (moDCs) and primary CD1c+ DCs. Notably, in contrast to other BV-associated microbiota, Prevotella timonensis enhanced uptake of HIV-1 by both moDCs and CD1c+ DCs and the increased uptake was independent of cellular HIV-1 (co-)receptors. Imaging flow cytometry analyses showed that HIV-1 did not co-localise with P. timonensis but was internalized into tetraspanin-positive compartments known to be involved in HIV-1 transmission. P. timonensis bacteria enhanced HIV-1 transmission by CD1c+ DCs, but not by moDCs, and the enhanced transmission was independent of viral infection. Our study strongly suggests that mucosal DC subsets have distinct functions in BV-associated HIV-1 susceptibility, and underscores the importance of early diagnosis and targeted treatment of vaginal dysbiosis to reduce the risk of HIV-1 acquisition.
© 2025 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.

Extensively drug-resistant tuberculosis (XDR-TB) is a public health concern as drug resistance is outpacing the drug development pipeline. Alternative immunotherapeutic approaches are needed. Peripheral blood mononuclear cells (PBMCs) were isolated from pre-XDR/XDR-TB (n = 25) patients and LTBI (n = 18) participants. Thereafter, monocytic-derived dendritic cells (mo-DCs) were co-cultured with M. tb antigens, with/without a maturation cocktail (interferon-γ, interferon-α, CD40L, IL-1β, and TLR3 and TLR7/8 agonists). Two peptide pools were evaluated: (i) an ECAT peptide pool (ESAT6, CFP10, Ag85B, and TB10.4 peptides) and (ii) a PE/PPE peptide pool. Sonicated lysate of the M. tb HN878 strain served as a control. Mo-DCs were assessed for DC maturation markers, Th1 cytokines, and the ability of the DC-primed PBMCs to restrict the growth of M. tb-infected monocyte-derived macrophages. In pre-XDR/XDR-TB, mo-DCs matured with M. tb antigens (ECAT or PE/PPE peptide pool, or HN878 lysate) + cocktail, compared to mo-DCs matured with M. tb antigens only, showed higher upregulation of co-stimulatory molecules and IL-12p70 (p < 0.001 for both comparisons). The matured mo-DCs had enhanced antigen-specific CD8+ T-cell responses to ESAT-6 (p = 0.05) and Ag85B (p = 0.03). Containment was higher with mo-DCs matured with the PE/PPE peptide pool cocktail versus mo-DCs matured with the PE/PPE peptide pool (p = 0.0002). Mo-DCs matured with the PE/PPE peptide pool + cocktail achieved better containment than the ECAT peptide pool + cocktail [50%, (IQR:39-75) versus 46%, (IQR:15-62); p = 0.02]. In patients with pre-XDR/XDR-TB, an effector response primed by mo-DCs matured with an ECAT or PE/PPE peptide pool + cocktail was capable of restricting the growth of M. tb in vitro.

Immunogenicity refers to the ability of a substance, such as a therapeutic drug, to elicit an immune response. While beneficial in vaccine development, undesirable immunogenicity can compromise the safety and efficacy of therapeutic proteins by inducing anti-drug antibodies (ADAs). These ADAs can reduce drug bioavailability and alter pharmacokinetics, necessitating comprehensive immunogenicity risk assessments starting at early stages of drug development. Given the complexity of immunogenicity, an integrated approach is essential, as no single assay can universally recapitulate the immune response leading to the formation of anti-drug antibodies.
To better understand the Dendritic Cell (DC) contribution to immunogenicity, we developed two flow cytometry-based assays: the DC internalization assay and the DC activation assay. Monocyte-derived dendritic cells (moDCs) were generated from peripheral blood mononuclear cells (PBMCs) and differentiated over a five-day period. The internalization assay measured the accumulation rate of therapeutic antibodies within moDCs, while the activation assay assessed the expression of DC activation markers such as CD40, CD80, CD86, CD83, and DC-SIGN (CD209). To characterize these two assays further, we used a set of marketed therapeutic antibodies.
The study highlights that moDCs differentiated for 5 days from freshly isolated monocytes were more prone to respond to external stimuli. The internalization assay has been shown to be highly sensitive to the molecule tested, allowing the use of only 4 donors to detect small but significant differences. We also demonstrated that therapeutic antibodies were efficiently taken up by moDCs, with a strong correlation with their peptide presentation on MHC-II. On the other hand, by monitoring DC activation through a limited set of activation markers including CD40, CD83, and DC-SIGN, the DC activation assay has the potential to compare a series of compounds. These two assays provide a more comprehensive understanding of DC function in the context of immunogenicity, highlighting the importance of both internalization and activation processes in ADA development.
The DC internalization and activation assays described here address key gaps in existing immunogenicity assessment methods by providing specific and reliable measures of DC function. The assays enhance our ability to pre-clinically evaluate the immunogenic potential of biotherapeutics, thereby improving their safety and efficacy. Future work should focus on further validating these assays and integrating them into a holistic immunogenicity risk assessment framework.
Copyright © 2024 Siegel, Padamsey, Bolender, Hargreaves, Fraidling, Ducret, Hartman, Looney, Bertinetti-Lapatki, Rohr, Hickling, Kraft and Marban-Doran.

AXL+ Siglec-6+ dendritic cells (ASDC) are novel myeloid DCs which can be subdivided into CD11c+ and CD123+ expressing subsets. We showed for the first time that these two ASDC subsets are present in inflamed human anogenital tissues where HIV transmission occurs. Their presence in inflamed tissues was supported by single cell RNA analysis of public databases of such tissues including psoriasis diseased skin and colorectal cancer. Almost all previous studies have examined ASDCs as a combined population. Our data revealed that the two ASDC subsets differ markedly in their functions when compared with each other and to pDCs. Relative to their cell functions, both subsets of blood ASDCs but not pDCs expressed co-stimulatory and maturation markers which were more prevalent on CD11c+ ASDCs, thus inducing more T cell proliferation and activation than their CD123+ counterparts. There was also a significant polarisation of naïve T cells by both ASDC subsets toward Th2, Th9, Th22, Th17 and Treg but less toward a Th1 phenotype. Furthermore, we investigated the expression of chemokine receptors that facilitate ASDCs and pDCs migration from blood to inflamed tissues, their HIV binding receptors, and their interactions with HIV and CD4 T cells. For HIV infection, within 2 hours of HIV exposure, CD11c+ ASDCs showed a trend in more viral transfer to T cells than CD123+ ASDCs and pDCs for first phase transfer. However, for second phase transfer, CD123+ ASDCs showed a trend in transferring more HIV than CD11c+ ASDCs and there was no viral transfer from pDCs. As anogenital inflammation is a prerequisite for HIV transmission, strategies to inhibit ASDC recruitment into inflamed tissues and their ability to transmit HIV to CD4 T cells should be considered.
Copyright: © 2024 Warner van Dijk et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Introduction: Exogeneous messenger ribonucleic acid (mRNA) can be used as therapeutic and preventive medication. However, during the enzymatic production process, commonly called in vitro transcription, by-products occur which can reduce the therapeutic efficacy of mRNA. One such by-product is double-stranded RNA (dsRNA). We therefore sought to limit the generation of dsRNA by-products during in vitro transcription. Materials and methods: In vitro transcription was performed with a DNA template including a poly(A)-tail-encoding region, dinucleotide or trinucleotide cap analogs for cotranscriptional capping, and relevant nucleoside triphosphates. Concentrations of UTP or modified UTP (m1ΨTP) and GTP were reduced and fed over the course of the reaction. mRNA was analyzed for dsRNA contamination, yield of the reaction, RNA integrity, and capping efficiency before translational activity was assessed. Results: Limiting the steady-state level of UTP or m1ΨTP during the enzymatic reaction reduced dsRNA formation, while not affecting mRNA yield or RNA integrity. Capping efficiency was optimized with the use of a combined GTP and UTP or m1ΨTP feed, while still reducing dsRNA formation. Lower dsRNA levels led to higher protein expression from the corresponding mRNAs. Discussion: Low steady-state concentrations of UTP and GTP, fed in combination over the course of the in vitro transcription reaction, produce mRNA with high capping and low levels of dsRNA formation, resulting in high levels of protein expression. This novel approach may render laborious purification steps to remove dsRNA unnecessary.
Copyright © 2023 Ziegenhals, Frieling, Wolf, Göbel, Koch, Lohmann, Baiersdörfer, Fesser, Sahin and Kuhn.