People living with HIV (PLWH) have an increased risk for developing tuberculosis after M. tuberculosis infection, despite anti-retroviral therapy (ART). To delineate the underlying mechanisms, we conducted single cell transcriptomics on bronchoalveolar lavage cells from PLWH on ART and HIV uninfected healthy controls infected with M. tuberculosis ex vivo. We identify an M1-like proinflammatory alveolar macrophage subset that sequentially acquires TNF signaling capacity in controls but not in PLWH. Cell-cell communication analyses reveal interactions between M1-like macrophages and effector memory T cells within TNF superfamily, chemokine, and costimulatory networks in the airways of controls. These interaction networks were lacking in PLWH infected with M. tuberculosis, where anti-inflammatory M2-like alveolar macrophages and T regulatory cells dominated along with dysregulated T cell signatures. Our data support a model in which impaired TNF-TNFR signaling, M2-like alveolar macrophages and aberrant macrophage-T cell crosstalk, lead to ineffective immunity to M. tuberculosis in PLWH on ART.
© 2025. The Author(s).

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.

Abstract People living with HIV (PLWH) have an increased risk for developing tuberculosis (TB) after infection with Mycobacterium tuberculosis (Mtb), despite anti-retroviral therapy (ART). To delineate the underlying mechanisms, we conducted single cell transcriptomics on bronchoalveolar lavage (BAL) cells from PLWH on ART and HIV uninfected healthy controls (HC) infected with Mtb ex vivo. We identified an M1-like proinflammatory alveolar macrophage (AM) subset that sequentially acquired TNF signaling capacity in HC but not in PLWH. Cell-cell communication analyses revealed robust interactions between M1-like AMs and effector memory T cells within TNF superfamily, chemokine, and costimulatory networks in the airways of HC. These interaction networks were lacking in PLWH infected with Mtb, where anti-inflammatory M2-like AMs and T regulatory cells dominated along with dysregulated T cell signatures. Our data support a model in which impaired TNF-TNFR signaling, and aberrant AM-T cell crosstalk, lead to ineffective immunity to Mtb in PLWH on ART.