Biology subjects

Fahlberg, M. D.

Publications and source records attributed to Fahlberg, M. D..

7 recordsLinked to original sources

bioRxiv2026

A 28-color panel for classical and non-classical T lymphocytes in decidua and PBMC in rhesus macaques

This 28-color panel was developed to identify classical and non-classical T lymphocytes in decidual leukocytes and peripheral blood mononuclear cells (PBMC) of pregnant rhesus macaques. By profiling these T lymphocytes, we can investigate how maternal immunity balances tolerance to fetal antigens with protection against vertically transmitted pathogens. The selected markers define memory populations and characterize tissue residency, activation, proliferation, cytotoxicity, trafficking, and exhaustion status. This panel also delineates B lymphocytes and NK cells to confirm expected frequencies. The utility of this panel is aimed at evaluating cellular immune correlates of protection against congenital infections at the maternal-fetal interface and PBMC in rhesus macaques.

immunology↗

bioRxiv2026

FlowSpot Enables Decentralized Phenotypic and Functional Cellular Immune Profiling from Dried Blood Spots

Expanding access to cellular immune analysis is essential for decentralized clinical care, clinical trials, and population-based research. However, current flow cytometry workflows require rapid processing of fresh blood, proximity to a centralized laboratory, and cold chain logistics. Although dried blood spots (DBS) have transformed decentralized molecular diagnostics, no comparable approach has enabled robust flow cytometric analysis of immune cells. Here, we present FlowSpot, a novel platform that enables recovery of leukocytes from DBS and preserves their immunophenotypic characteristics, allowing downstream flow cytometric analysis following ambient-temperature storage and shipment. FlowSpot recovers intact leukocytes while preserving immune cell subset frequencies with strong concordance to fresh whole blood. We demonstrate its clinical utility by enabling remote CD4 T cell immunophenotyping in people living with HIV, showing high agreement with routine clinical measurements across a broad range of CD4 T cell frequencies. Beyond cellular phenotyping, FlowSpot extends immune monitoring to functional profiling by enabling detection of intracellular cytokine responses, including IFN{gamma}, IL-2, and TNF production by CD4 and CD8 T cells following ex vivo PMA/ionomycin stimulation. By overcoming a longstanding barrier to leukocyte recovery from DBS, FlowSpot extends flow cytometry beyond specialized laboratories, expanding access to cellular immune analysis for clinical care, decentralized clinical trials, and population-scale immunology.

immunology↗

bioRxiv2025

Differentiation stage-specific use of cap-independent and cap-dependent translation initiation in hematopoiesis

Cell stress can increase the use of m7G-cap-independent, IRES-mediated translation initiation relative to cap-dependent translation (IRES/Cap). Reporters that quantify IRES/Cap have demonstrated differential activity across cultured cell types and stress conditions. By generating an IRES/Cap reporter mouse, we were able to systematically evaluate IRES/Cap across distinct tissues and cell types during physiological stresses and lineage commitment. Caloric stress invoked the expected boost in IRES/Cap translation regardless of differentiation state, but unexpectedly IRES/Cap progressively increased during hematopoietic and epithelial (hair follicle) differentiation under normal, homeostatic conditions. This was independent of total protein output or cell cycle. Even within cells of a given differentiation state, cells with lower relative-IRES utilization had markedly higher multipotent capability in vivo. The RNA processing protein PTBP1 is a mediator of this translation initiation preference. Therefore, low IRES/Cap is a signature of high stemness and suggests modulation of translation initiation participates in cell differentiation state.

developmental biology↗

bioRxiv2024

Overcoming fixation and permeabilization challenges in flow cytometry by optical barcoding and multi-pass acquisition

The fixation and permeabilization of cells are essential for labeling intracellular biomarkers in flow cytometry. However, these chemical treatments often alter fragile targets, such as cell surface and fluorescent proteins, and can destroy chemically-sensitive fluorescent labels. This reduces measurement accuracy and introduces compromises into sample workflows, leading to losses in data quality. Here, we demonstrate a novel multi-pass flow cytometry approach to address this long-standing problem. Our technique utilizes individual cell barcoding with laser particles, enabling sequential analysis of the same cells with single-cell resolution maintained. Chemically-fragile protein markers and their fluorochrome conjugates are measured prior to destructive sample processing and adjoined to subsequent measurements of intracellular markers after fixation and permeabilization. We demonstrate the effectiveness of our technique in accurately measuring intracellular fluorescent proteins and methanol-sensitive antigens and fluorophores, along with various surface and intracellular markers. This approach significantly enhances assay flexibility, enabling accurate and comprehensive cell analysis without the constraints of conventional one-time measurement flow cytometry. This innovation paves new avenues in flow cytometry for a wide range of applications in immuno-oncology, stem cell research, and cell biology.

bioengineering↗

bioRxiv2022

Laser particle barcoding for multi-pass high-dimensional flow cytometry

Flow cytometry is a standard technology in life science and clinical laboratories used to characterize the phenotypes and functional status of cells, especially immune cells. Recent advances in immunology and immuno-oncology as well as drug and vaccine discovery have increased the demand to measure more parameters. However, the overlap of fluorophore emission spectra and one-time measurement nature of flow cytometry are major barriers to meeting the need. Here, we present multi-pass flow cytometry, in which cells are tracked and measured repeatedly through barcoding with infrared laser-emitting microparticles. We demonstrate the benefits of this approach on several pertinent assays with human peripheral blood mononuclear cells (PBMCs). First, we demonstrate unprecedented time-resolved flow characterization of T cells before and after stimulation. Second, we show 33-marker deep immunophenotyping of PBMCs, analyzing the same cells in 3 back-to-back cycles. This workflow allowed us to use only 10-13 fluorophores in each cycle, significantly reducing spectral spillover and simplifying panel design. Our results open a new avenue in multi-dimensional single-cell analysis based on optical barcoding of individual cells.

bioengineering↗

bioRxiv2021

Adjuvant and immunomodulatory potential of Natural Killer T (NKT) activation by NKTT320

Invariant natural killer T-lymphocytes (iNKT) are unique immunomodulatory innate T-cells with an invariant TCR recognizing glycolipids presented on MHC class-I-like CD1d molecules. Activated iNKT rapidly secrete pro-and anti-inflammatory cytokines, potentiate innate and adaptive immunity, and modulate inflammation. Here, we report the effects of in vivo iNKT activation by a novel humanized monoclonal antibody, NKTT320, that binds to the invariant region of the iNKT TCR. NKTT320 led to rapid iNKT activation, increased polyfunctionality, and elevation of multiple plasma analytes within 24 hours of administration. Flow cytometry and RNA-Seq confirmed downstream activation of multiple immune subsets, enrichment of JAK/STAT and PI3K/AKT pathway genes, and upregulation of inflammation-modulating genes CMKLR1, ARG2 and NLRP12. NKTT320 also induced iNKT trafficking to adipose tissue and did not cause iNKT anergy. Our data indicate that NKTT320 has a sustained effect on in vivo iNKT activation, potentiation of innate and adaptive immunity, and resolution of inflammation, which supports its future use as an immunotherapeutic and vaccine adjuvant. SummaryiNKTs are known immunomodulatory cells whose activation is a potential target for immunotherapies and use as an adjuvant. Here we report the potential utility of in vivo iNKT activation using the novel humanized monoclonal antibody NKTT320 for this purpose.

immunology↗

bioRxiv2020

Cellular events of acute, resolving or progressive COVID-19 in SARS-CoV-2 infected non-human primates

We investigated the immune events following SARS-CoV-2 infection, from the acute inflammatory state up to four weeks post infection, in non-human primates (NHP) with heterogeneous pulmonary pathology. The acute phase was characterized by a robust and rapid migration of monocytes expressing CD16 from the blood and concomitant increase in CD16+ macrophages in the lungs. We identified two subsets of interstitial macrophages (HLA-DR+ CD206-), a transitional CD11c+ CD16+ cell population that was directly associated with IL-6 levels in plasma, and one long lasting CD11b+ CD16+ cell population. Strikingly, levels of monocytes were a correlate of viral replication in bronchial brushes and we discovered TARC (CCL17) as a new potential mediator of myeloid recruitment to the lungs. Worse disease outcomes were associated with high levels of cell infiltration in lungs including CD11b+ CD16hi macrophages and CD11b+ neutrophils. Accumulation of macrophages was long-lasting and detectable even in animals with mild or no signs of disease. Interestingly, animals with anti-inflammatory responses including high IL-10:IL-6 and kynurenine to tryptophan ratios had less signs of disease. Our results unravel cellular mechanisms of COVID-19 and suggest that NHP may be appropriate models to test immune therapies.

immunology↗