Biology subjects

Mbiribindi, B.

Publications and source records attributed to Mbiribindi, B..

3 recordsLinked to original sources

bioRxiv2026

Practical quantification of immunohistochemistry antigen concentrations and reaction-diffusion parameters

The immunohistochemistry (IHC) methods widely used in diagnostic medicine and biomedical research are kinetically complex reaction-diffusion processes that, ideally, produce stain intensities correlated with the local antigen concentration. Yet after 75 years of use, practical theoretical tools to rigorously plan and interpret IHC experiments are still lacking. Because modeling the reactions requires time-consuming computer simulation, impractical for regular use, most protocols are optimized empirically, without detailed knowledge of the reaction rates and antigen-antibody equilibria. The resulting stain intensities can be calibrated against standards with known antigen abundance, but they are typically not interpretable in terms of chemical antigen concentrations. To address these limitations, we developed a fast interpolation method to model reaction-diffusion behavior, and experimental methods to characterize IHC kinetic parameters in formalin-fixed paraffin-embedded (FFPE) samples. Used together, these allow experimental measurement of both the chemical concentration of antigen in the sample and the reaction-diffusion parameters consistent with the assay results. Results show 1) direct immunofluorescent detection has low nanomolar sensitivity with >1000-fold dynamic range, and 2) antibody diffusion rates in FFPE samples can be >1000-fold slower than in aqueous solutions, producing diffusion-limited conditions in which the IHC reaction time course may depend on the sample antigen concentration. Awareness of these details is necessary to avoid potential underestimation of both the absolute and relative antigen concentrations in different samples that may occur if staining is stopped before reaching equilibrium. Software tools are provided to allow users to rapidly model IHC reaction time courses and to fit experimental time course data with candidate reaction parameters. The principles described here apply equally to other tissue-based "spatial omics" analyses and should be considered when designing and interpreting experiments requiring any macromolecule to diffuse into and react in a tissue section. SIGNIFICANCEThe theoretical and experimental framework described here advances IHC staining from a qualitative or semi-quantitative method towards a more rigorously quantitative assay. The practical ability to predict IHC reaction kinetics and fit reaction parameters to experimental data has the potential to advance IHC applications in diagnostic medicine and biomedical research in three ways: 1) interpretation of experimental and diagnostic samples stained under different conditions can be more objective, facilitating comparison of results from different protocols and different laboratories; 2) IHC staining can be interpreted as molar chemical antigen-antibody concentrations calculated from the reaction parameters measured in the studied sample; 3) the correlation between antigen concentration and biological behavior can be examined more reliably. Practical software tools are provided.

pathology↗

bioRxiv2021

Human IL-10-producing B cells have diverse states induced from multiple B cell subsets

Regulatory B cells (Bregs) can suppress immune responses through the secretion of IL-10 and other anti-inflammatory cytokines. This immunomodulatory capacity holds therapeutic potential, yet a definitional immunophenotype for enumeration and prospective isolation of B cells capable of IL-10 production remains elusive. We therefore applied mass cytometry to simultaneously quantify cytokine production and immunophenotype in human peripheral B cells across a range of stimulatory conditions and timepoints. While multiple B cell subsets produced IL-10, no phenotype uniquely identified IL-10+ B cells and a significant portion of IL-10+ B cells co-expressed the proinflammatory cytokines IL-6 and TNF. Despite this heterogeneity, we found operationally tolerant liver transplant recipients had a unique enrichment of IL-10+, but not TNF+ or IL-6+, B cells as compared to transplant recipients receiving immunosuppression. Thus, human IL-10-producing B cells constitute an induced, transient state arising from a diversity of B cell subsets that may contribute to maintenance of immune homeostasis.

immunology↗

bioRxiv2020

A DNA vaccine strategy to activate natural killer cells for therapeutic targeting of XPO1-positive tumors

Natural killer (NK) cells are key components of the immune response to viral infections and cancer. Their functions are controlled by activating and inhibitory killer-cell immunoglobulin-like receptors (KIR) which have MHC class I ligands. KIR2DS2 is an activating KIR, that binds conserved viral peptides in the context of HLA-C and has been associated with protective responses to both cancer and viral infections. We sought to investigate whether NK cells can be specifically activated in a peptide:MHC dependent manner to generate functional immune responses as a potential immunotherapeutic strategy. We developed a peptide-based KIR targeting DNA vaccine. Immunizing KIR-Tg mice with the vaccine construct generated in vivo peptide-specific activation of KIR2DS2-positive NK cells leading to canonical and cross-reactive peptide specific immune responses in vitro, and also in vivo inhibition of tumor growth. Using immunopeptidomics we identified that the nuclear export protein XPO1, which has been associated with a poor prognosis in many different human cancers, furnishes an HLA-C restricted cancer-associated peptide ligand for KIR2DS2-positive NK cells. We thus define a novel strategy to activate KIR in a peptide-specific manner and identify a rationale for its use in cancer immunotherapy. Significance statementNatural killer (NK) cells are known to have important roles in determining the outcomes of viral infections and cancer. The killer cell immunoglobulin-like receptors (KIR), and in particular the activating receptor KIR2DS2, have been associated with the outcome of a number of different human cancers. Specific activation of NK cells through KIR2DS2 is challenging because it shares high (>98%) sequence homology with related inhibitory KIR. We have used a peptide:MHC targeting strategy to activate NK cells through KIR2DS2 and identified a novel cancer-associated ligand for this receptor. The work provides a proof-of-concept for targeting NK cells through activating KIR as a cancer immunotherapy strategy.

immunology↗