Biology subjects

Krueger, J.

Publications and source records attributed to Krueger, J..

11 recordsLinked to original sources

bioRxiv2026

Multiplex engineering of rhesus macaque NK cells enhances homing to sites of HIV replication in B cell follicles.

One barrier to developing an HIV-1 cure is viral reservoirs persisting within B cell follicles of lymphatic tissues, partly due to failure of HIV-specific cytotoxic cells to express the follicular-homing receptor CXCR5. Our group explores CAR cell therapies which also express CXCR5 as a potential cure strategy for HIV. Although previous studies have mostly explored CAR T cell therapies, CAR NK cells may be an attractive alternative as they can be used in allogeneic settings and are naturally cytotoxic towards HIV-infected cells. Here, we developed a novel and innovative multiplex engineering method for rhesus macaque NK cells to create virus-specific CAR NK cells multiplexed (MP) with CAR/CXCR5/IL-15/PD-1 KO/transient-CCR7. We first evaluated MP NK cells in vitro for functionality. MP NK cells were then infused into one chronically SIV-infected rhesus macaque to observe tolerance and localization of therapeutic cells. Finally, we performed a larger primate study in which SIV-infected rhesus macaques were infused with two doses of MP NK cells to study long-term localization, safety, and efficacy. In vitro, MP NK cells were expanded to clinically relevant numbers, migrated to chemokine signaling, and secreted cytotoxic cytokines in response to SIV-Env-expressing cells. In the preliminary rhesus macaque study, the therapy caused no adverse reactions, and CAR+ NK cells localized to sites of SIV replication within the spleen and lymph nodes. In the larger primate study, two doses of MP NK cells at 1.2 x 108 cells/kg were safe and increased the levels of NK cells and CAR+ NK cells found within lymphatic tissues. Importantly, the CAR+ NK cells detected in lymph nodes were predominantly CCR7+, demonstrating the importance of CCR7 and CXCR5 in combination for migration to SIV viral reservoirs in follicles of lymphatic tissues. This study is the first to demonstrate this type of complexity and combination of engineering techniques in NK cells. With further optimization, these techniques could lead to the development of novel NK cell therapies to treat HIV and other diseases.

bioengineering↗

bioRxiv2026

Calcium-binding protein expression alone is insufficient to identify and classify GABAergic neurons in macaque cortex

Understanding neuron subclasses and their functional consequences can contribute to understanding brain circuits. A scheme long used to classify GABAergic neurons in the neocortex is based on expression of three calcium-binding proteins (CBPs): parvalbumin (PV), calbindin D-28K (CB), and calretinin (CR). Because CB and CR are frequently co-expressed by individual neurons in rodents, this scheme has been replaced by one based on PV and two signaling peptides: somatostatin (SST) and vasoactive intestinal peptide (VIP). In macaques, however, CBPs are generally not co-expressed, and so their use has persisted despite suggestions that the underlying populations are not, in fact, entirely GABAergic. We set out to quantitatively evaluate CBPs as a classification scheme for GABAergic neurons in early and mid-level visual regions in macaque cortex. Combining immunohistochemistry and in situ hybridization, we find that up to half of neurons expressing CBPs are likely not GABAergic. Furthermore, contrary to what has been previously suggested, the GABAergic subpopulations cannot be distinguished based on staining intensity. Thus, the CBP-based classification scheme is not valid, at least as it has traditionally been used. Instead, we find support for co-labeling CB and CR neurons with SST and VIP, an approach that can identify GABAergic subpopulations within the CBP classes; or simply adopting the PV/SST/VIP scheme. We discuss the functional implications of expressing these various cell type markers, and how consideration of marker functions can support proper selection of a classification scheme for a given experimental purpose. Significance StatementThe findings of this study challenge the currently dominant classification scheme for GABAergic neurons in macaque cortex, a scheme based on expression of the calcium-binding proteins parvalbumin, calbindin D-28K, and calretinin. Specifically, we show that a large proportion of neurons that express protein or mRNA for these calcium-binding proteins are likely not GABAergic. We go on to consider alternative approaches, discussing under what circumstances each might be useful.

neuroscience↗

bioRxiv2025

Single Cell Analysis of pBMCs of Psoriasis patients reveals distinct CD4+ T cell phenotypes associated with response to IL-23 blockade.

The use of IL-23 inhibitors (IL-23i) for psoriatic diseases has resulted in significant improvement in disease symptoms for many patients. The extent of disease improvement following IL-23 blockade varies across patients with psoriasis; however, the immunologic factors associated with a good or poor response to IL-23 blockade remain unclear. Here, we utilized peripheral blood mononuclear cells (pBMCs) collected through the MINIMA clinical trial (NCT04271540) and applied single-cell RNA sequencing to profile circulating immune populations from 27 patients with psoriasis or psoriatic arthritis, aiming to identify cellular features associated with a response to IL-23 blockade. We identified populations of CD4+ T cells whose abundance in circulation before treatment was associated with improved skin disease following IL-23i treatment. Circulating CD4+ T cells that demonstrate transcriptomic features of Th1-like cells were associated with better psoriasis skin improvement and had transcriptomic signatures resembling T cells identified in psoriasis lesional skin. Further, decrease in levels of IL-17A and IFN{gamma} in serum each correlated with improvement in PASI levels. These results suggest that immune cell features detectable in blood may be informative in identifying patients with psoriasis who are likely to have a robust clinical response to IL-23 blockade.

immunology↗

bioRxiv2025

ProteoParc: A tool to generate protein reference databases for ancient and non-model organisms

Over the last few years, the increasing interest in analysing the proteome of extinct and non-model organisms has generated a new field of research expanding the scope of proteomics. The lack of curated databases and/or molecular data from these organisms forces researchers to manually search in different public repositories for related protein sequences, either for MS/MS peptide identification or ZooMS marker annotation. This can lead to format incongruences and hinder reproducibility between studies. To address this issue, we introduce ProteoParc, a user-friendly software that generates reference databases by systematically downloading and processing protein sequences from the most widely used public repositories. The pipelines output is a non-redundant protein database, formatted to be interpreted by typical peptide identification software. Moreover, the user can adjust the database dimension and composition by applying different criteria to include only a certain number of genes or species. Thus, ProteoParc is an easy and fast, custom-made bioinformatic tool useful for future paleoproteomics analysis in ancient samples related to understudied organisms.

bioinformatics↗

bioRxiv2024

Efficient multiplex non-viral engineering and expansion of polyclonal γδ CAR-T cells for immunotherapy

Gamma delta ({gamma}{delta}) T cells are defined by their unique ability to recognize a limited repertoire of non-peptide, non-MHC-associated antigens on transformed and pathogen-infected cells. In addition to their lack of alloreactivity, {gamma}{delta} T cells exhibit properties distinct from other lymphocyte subsets, prompting significant interest in their development as an off-the-shelf cellular immunotherapeutic. However, their low abundance in circulation, heterogeneity, limited methods for ex vivo expansion, and under-developed methodologies for genetic modification have hindered basic study and clinical application of {gamma}{delta} T cells. Here, we implement a feeder-free, scalable approach for ex vivo manufacture of polyclonal, non-virally modified, gene edited chimeric antigen receptor (CAR)-{gamma}{delta} T cells in support of therapeutic application. Engineered CAR-{gamma}{delta} T cells demonstrate high function in vitro and and in vivo. Longitudinal in vivo pharmacokinetic profiling of adoptively transferred polyclonal CAR-{gamma}{delta} T cells uncover subset-specific responses to IL-15 cytokine armoring and multiplex base editing. Our results present a robust platform for genetic modification of polyclonal CAR-{gamma}{delta} T cells and present unique opportunities to further define synergy and the contribution of discrete, engineered CAR-{gamma}{delta} T cell subsets to therapeutic efficacy in vivo.

immunology↗

bioRxiv2024

Phylogenetic signal in primate tooth enamel proteins and its relevance for paleoproteomics

Ancient tooth enamel, and to some extent dentin and bone, contain characteristic peptides that persist for long periods of time. In particular, peptides from the enamel proteome (enamelome) have been used to reconstruct the phylogenetic relationships of fossil specimens and to estimate divergence times. However, the enamelome is based on only about 10 genes, whose protein products undergo fragmentation post mortem. Moreover, some of the enamelome genes are paralogous or may coevolve. This raises the question as to whether the enamelome provides enough information for reliable phylogenetic inference. We address these considerations on a selection of enamel-associated proteins that has been computationally predicted from genomic data from 232 primate species. We created multiple sequence alignments (MSAs) for each protein and estimated the evolutionary rate for each site and examined which sites overlap with the parts of the protein sequences that are typically isolated from fossils. Based on this, we simulated ancient data with different degrees of sequence fragmentation, followed by phylogenetic analysis. We compared these trees to a reference species tree. Up to a degree of fragmentation that is similar to that of fossil samples from 1-2 million years ago, the phylogenetic placements of most nodes at family level are consistent with the reference species tree. We found that the composition of the proteome influences the phylogenetic placement of Tarsiiformes. For the inference of molecular phylogenies based on paleoproteomic data, we recommend characterizing the evolution of the proteomes from the closest extant relatives to maximize the reliability of phylogenetic inference.

evolutionary biology↗

bioRxiv2023

Enamel proteins reveal biological sex and genetic variability within southern African Paranthropus

The evolutionary relationships among extinct African hominin taxa are highly debated and largely unresolved, due in part to a lack of molecular data. Even within taxa, it is not always clear, based on morphology alone, whether ranges of variation are due to sexual dimorphism versus potentially undescribed taxonomic diversity. For Paranthropus robustus, a Pleistocene hominin found only in South Africa, both phylogenetic relationships to other taxa 1,2 and the nature of intraspecific variation 3-6 are still disputed. Here we report the mass spectrometric (MS) sequencing of enamel proteomes from four ca. 2 million year (Ma) old dental specimens attributed morphologically to P. robustus, from the site of Swartkrans. The identification of AMELY-specific peptides and semi-quantitative MS data analysis enabled us to determine the biological sex of all the specimens. Our combined molecular and morphometric data also provide compelling evidence of a significant degree of variation within southern African Paranthropus, as previously suggested based on morphology alone 6. Finally, the molecular data also confirm the taxonomic placement of Paranthropus within the hominin clade. This study demonstrates the feasibility of recovering informative Early Pleistocene hominin enamel proteins from Africa. Crucially, it also shows how the analysis of these proteins can contribute to understanding whether hominin morphological variation is due to sexual dimorphism or to taxonomic differences. We anticipate that this approach can be widely applied to geologically-comparable sites within South Africa, and possibly more broadly across the continent.

evolutionary biology↗

bioRxiv2021

Spike mutation T403R allows bat coronavirus RaTG13 to use human ACE2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 pandemic, most likely emerged from bats1. A prerequisite for this devastating zoonosis was the ability of the SARS-CoV-2 Spike (S) glycoprotein to use human angiotensin-converting enzyme 2 (ACE2) for viral entry. Although the S protein of the closest related bat virus, RaTG13, shows high similarity to the SARS-CoV-2 S protein it does not efficiently interact with the human ACE2 receptor2. Here, we show that a single T403R mutation allows the RaTG13 S to utilize the human ACE2 receptor for infection of human cells and intestinal organoids. Conversely, mutation of R403T in the SARS-CoV-2 S significantly reduced ACE2-mediated virus infection. The S protein of SARS-CoV-1 that also uses human ACE2 also contains a positive residue (K) at this position, while the S proteins of CoVs utilizing other receptors vary at this location. Our results indicate that the presence of a positively charged amino acid at position 403 in the S protein is critical for efficient utilization of human ACE2. This finding could help to predict the zoonotic potential of animal coronaviruses.

microbiology↗

bioRxiv2020

Enhancement of synaptic AMPA receptors depends mutually on Src and PSD-95

Synaptic incorporation and removal of AMPA receptors is highly regulated to modulate the strength of synaptic transmission for long-term synaptic plasticity during brain development and associative learning. PSD-932 and PSD-95, two paralogs of the DLG-MAGUK protein family of signaling scaffolds govern the synaptic incorporation and stabilization of AMPA receptors opposingly, with PSD-95 promoting and PSD-932 inhibiting it. The associated signaling mechanisms that control the synaptic incorporation and stabilization remain elusive. Here, we used domain swapping between the antagonizing signaling scaffolds to identify the protein motifs responsible for enhancing synaptic AMPA receptors and the associated signaling protein. We narrowed down multiple motifs in the N-terminal domain that are principally responsible for governing the enhancement by Src. Specific activation and inhibiting peptides revealed continuous activity of Src. Together, the results depict a mutual dependence of Src and PSD-95 in enhancing and maintaining synaptic AMPA receptors.

neuroscience↗

bioRxiv2020

IFITM proteins promote SARS-CoV-2 infection in human lung cells

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) are thought to restrict numerous viral pathogens including severe acute respiratory syndrome coronaviruses (SARS-CoVs). However, most evidence comes from single-round pseudovirus infection studies of cells that overexpress IFITMs. Here, we verified that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. Strikingly, however, endogenous IFITM expression was essential for efficient infection of genuine SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral entry. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. Intriguingly, IFITM-derived peptides and targeting antibodies inhibited SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are important cofactors for SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and suitable targets for therapeutic approaches.

microbiology↗

bioRxiv2020

Remdesivir but not famotidine inhibits SARS-CoV-2 replication in human pluripotent stem cell-derived intestinal organoids

Gastrointestinal symptoms in COVID-19 are associated with prolonged symptoms and increased severity. We employed human intestinal organoids derived from pluripotent stem cells (PSC-HIOs) to analyze SARS-CoV-2 pathogenesis and to validate efficacy of specific drugs in the gut. Certain, but not all cell types in PSC-HIOs express SARS-CoV-2 entry factors ACE2 and TMPRSS2, rendering them susceptible to SARS-CoV-2 infection. Remdesivir, a promising drug to treat COVID-19, effectively suppressed SARS-CoV-2 infection of PSC-HIOs. In contrast, the histamine-2-blocker famotidine showed no effect. Thus, PSC-HIOs provide an interesting platform to study SARS-CoV-2 infection and to identify or validate drugs.

microbiology↗