Biology subjects

Kosaka, Y.

Publications and source records attributed to Kosaka, Y..

7 recordsLinked to original sources

bioRxiv2026

Engineering antigen-driven co-stimulation and T helper cell activity into TCR-T cells with CD8-41BB fusion receptors enhances anti-tumor activity

1Adoptive cell therapy using tumor antigen-targeting T cell receptors (TCRs) offers a compelling approach to treat both hematological cancers and solid tumors due to broad antigen accessibility and the ability to target cancer-specific neoantigens. However, unlike clinically validated second generation CAR-T cells bearing built-in co-stimulatory signaling modules (i.e. 41BB or CD28), TCR-T cells receive little to no co-stimulation within most tumor microenvironments leading to attenuated cellular responses. Additionally, CD4+ TCR-T cells engineered to express HLA-Class I restricted TCRs possess minimal T-helper cell activity and thus do not effectively mobilize CD8+ TCR-T cells or host anti-tumor immune responses. To address these limitations, we used CRISPR-Cas9 to engineer TCR-T cells with targeted integration of chimeric CD8 constructs containing intracellular co-stimulatory domains. We found that expression of wild-type CD8{beta}, but not CD8, could promote CD4+ T cell activities in HLA-Class I restricted TCR-T cells. However, this was insufficient to drive durable anti-tumor responses in challenging tumor mouse models when using a high-affinity WT1-directed TCR. To address this, several CD8 co-stimulatory fusion constructs containing CD28 or 41BB intracellular domains were designed and screened, identifying two CD8-41BB based chimeras that substantially increased TCR-T cell activity relative to wild-type CD8{beta}. WT1-TCR-T cells co-expressing the CD8-41BB fusions demonstrated not only enhanced CD4+ activity including strong and polarized Th1-type cytokine secretion, but also enhanced the proliferation, cytokine release, and cytotoxicity of CD8+ CTLs. Remarkably, when combined with TGFBR2 gene disruption, WT1-TCR-T cells co-expressing CD8-41BB receptors were able to completely regress established cell line-derived ovarian tumors, showed robust in vivo expansion and persistence, and provided long-term protection from tumor rechallenge. Importantly, the specificity profile of the WT1-TCR including its HLA-A*02:01 restriction and WT1 peptide recognition motif was preserved upon expression of CD8-41BB. To simplify cell engineering processes for clinical applications, we configured a homology directed repair (HDR) cassette to allow for efficient CRISPR-Cas9-based insertion of both the TCR and CD8-41BB transgenes in the TRAC locus in a single step with >80% efficiency. Lastly, the enhanced activity conferred by CD8-41BB expression was validated with a second clinically relevant TCR targeting PRAME, suggesting this platform can be a universal approach for enhancing the therapeutic potential of TCR-based cell therapies.

synthetic biology↗

bioRxiv2026

Azacytidine restores T cell function in AML by modulating DNA methylation

AML is an aggressive blood cancer associated with poor clinical outcomes. Chemotherapy remains the standard of treatment, but unfortunately relapse is very common, highlighting the need for alternative therapies. T cell dysfunction and exhaustion are prominent in AML and may represent a barrier to effective immunotherapy yet remains poorly studied in AML. DNA methylation is a major driver of T cell exhaustion and inhibition of de novo methylation can block exhaustion and restore T cell function in chronic viral infections and other cancers but is understudied in AML. Here, we investigated the impact of azacytidine (Aza), an FDA-approved hypomethylating agent, on T cell exhaustion in AML. Using a spontaneous AML mouse model and samples from patients with AML, we found that Aza treatment modulates T cell function. In vivo Aza-treatment of AML-bearing mice decreased tumor burden and reshaped CD8+ T cell states, with increases in frequencies of memory subsets and decreases in regulatory T cells (Tregs). Functionally, Aza treatment overcame the impaired proliferation displayed by both CD4 and CD8+ T cells in our model. DNA methylation sequencing of T cells after Aza treatment revealed hypomethylation and increased expression of stem-like precursor gene TCF7 and E2F2, a regulator of cell cycle progression and proliferation. Similar changes in phenotypes were observed in cultures of AML patient samples treated with Aza. Collectively, we show that Aza remodels epigenetic and functional states in AML and has the potential to reverse T cell exhaustion, with enhanced memory and proliferation capacity. Our work generates a mechanistic framework that provides rationale of combining hypomethylating agents with T cell-based immunotherapies in this lethal disease. Data Sharing StatementRRBS data is available in GEO under the accession number GSE328721. For original data please contact Dr. Evan F. Lind. Key PointsAzacytidine mediated epigenetic modulation can alleviate T cell exhaustion in AML Translational RelevanceImmune therapy has shown limited efficacy in AML, despite increasing evidence of T cell dysfunction in this malignancy. Azacytidine (Aza) is an FDA approved drug for AML, but patients develop therapy resistance and relapse. Studies have mainly focused on Azas tumor intrinsic effects. In this study, we investigated the impact of Aza on immune function, especially T cell exhaustion in AML, since exhaustion is a major mechanism of disease resistance. We demonstrated that Aza can modulate T cell phenotype and restore T cell proliferation. Mechanistically, Aza induces epigenetic reprogramming in T cells and increases the expression of a stem-like precursor marker, TCF7. By shifting the focus on T cell biology, our study provides a rationale for combining Aza with other immunotherapies that can enhance durable immune responses in this malignancy.

immunology↗

bioRxiv2023

Leukemic mutation FLT3-ITD is retained in dendritic cells and disrupts their homeostasis leading to expanded Th17 frequency

Dendritic cells (DC) are mediators of adaptive immune responses to pathogens and tumors. DC development is determined by signaling through the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3) in bone marrow myeloid progenitors. Recently the naming conventions for DC phenotypes have been updated to distinguish between "Conventional" DCs (cDCs) and plasmacytoid DCs (pDCs). Activating mutations of FLT3, including Internal Tandem Duplication (FLT3-ITD), are associated with poor prognosis for leukemia patients. To date, there is little information on the effects of FLT3-ITD in DC biology. We examined the cDC phenotype and frequency in bone marrow aspirates from patients with acute myeloid leukemia (AML) to understand the changes to cDCs associated with FLT3-ITD. When compared to healthy donor (HD) we found that a subset of FLT3-ITD+ AML patient samples have overrepresented populations of cDCs and disrupted phenotypes. Using a mouse model of FLT3-ITD+ AML, we found that cDCs were increased in percentage and number compared to control wild-type (WT) mice. Single cell RNA-seq identified FLT3-ITD+ cDCs as skewed towards a cDC2 T-bet-phenotype, previously shown to promote Th17 T cells. We assessed the phenotypes of CD4+ T cells in the AML mice and found significant enrichment of both Treg and Th17 CD4+ T cells. Furthermore, co-culture of AML mouse- derived DCs and naive OT-II cells preferentially skewed T cells into a Th17 phenotype. Together, our data suggests that FLT3-ITD+ leukemia-associated cDCs polarize CD4+ T cells into Th17 subsets, a population that has been shown to be negatively associated with survival in solid tumor contexts. This illustrates the complex tumor microenvironment of AML and highlights the need for further investigation into the effects of FLT3-ITD mutations on DC phenotypes.

immunology↗

bioRxiv2022

Reconstitution of ribosome self-replication outside a living cell

Ribosome biogenesis, a recursive process of pre-existing ribosomes self-replicating nascent ones, is pivotal in the self-replication of life. In Escherichia coli, three ribosomal RNAs (rRNAs) are transcribed, and 54 ribosomal proteins (r-proteins) are synthesized by pre-existing ribosomes as structural components1, 2. They are cotranscriptionally assembled in a cooperative hierarchy under the support of [~]100 accessory factors1-3. The reconstitution of ribosome biogenesis outside a living cell is an essential goal to understand the self-replication of life. However, this goal could not have been achieved so far due to its complexity. Here, we report the successful in vitro reconstitution of the entire ribosome biogenesis process. We hypothesized that mimicking in vivo ribosome biogenesis1-6 could result in in vitro ribosome biogenesis. Specifically, we found that coactivating the transcription of an rRNA operon, as well as the transcription and translation of 54 r-protein genes encoding r-proteins, and the coordinated ribosomal assembly in a cytoplasm-mimicking reaction solution, resulted in highly efficient in vitro reconstitution of ribosome biogenesis. Our achievement represents a critical step toward revealing fundamental principles underlying the self-replication of life and creating self-replicating artificial cells7. We also succeeded in engineering rRNA and r-proteins by only adding mutant ribosomal genes in the reaction, enabling high-throughput and unconstrained creation of artificial ribosomes with altered or enhanced functionality8-12.

synthetic biology↗

bioRxiv2021

Morphological variation and phylogeny of Karenia selliformis (Gymnodiniales, Dinophyceae) in an intensive cold-water algal bloom in eastern Hokkaido, Japan in September-November 2021

Harmful algal blooms responsible for mass mortalities of marine organisms have so far been rare in Hokkaido, northern Japan, although fish killing blooms have been frequently reported from western Japanese coasts. In September-November 2021, a huge and prolonged cold-water bloom occurred along the Pacific coast of eastern Hokkaido, Japan, and was associated with intensive mortalities of sea urchin, fish, octopus, shellfish, etc. In this study, morphology and phylogeny of the dominant and co-occurred unarmored dinoflagellates of the Kareniaceae in the bloom were examined by using light microscopy, scanning electron microscopy and molecular phylogeny inferred from ITS and LSU rDNA (D1-D3) sequences. Morphological observation and molecular phylogeny showed that the dominant species was Karenia selliformis, with co-occurrences of other kareniacean dinoflagellates, Kr. longicanalis, Kr. mikimotoi, Karlodinium sp., Takayama cf. acrotrocha, Takayama tuberculata and Takayama sp. The typical cell forms of K. selliformis in the bloom were discoid, dorsoventrally flattened, and larger than the cell sizes in previous reports, 35.3-43.6 (39.4{+/-}2.1) m in length. Transparent cells of Kr. selliformis lacking or having several shrunken chloroplasts and oil droplets were also found. Cells of Kr. selliformis had morphological variation, but the species could be distinguished from other co-occurred Karenia species by its numerous (46-105) and small granular (2.9-4.6 m in diameter) chloroplasts and the nucleus positioned in the hypocone. Cell density of Kr. selliformis exceeding 100 cells/mL was recorded in the range of temperature 9.8-17.6{degrees}C. The rDNA sequences determined from Kr. selliformis in the blooms of Hokkaido, Japan in 2021 were identical to those from another bloom in Kamchatka, Russia in 2020. HighlightsO_LIA marine fauna-destructive harmful algal bloom in the Pacific coast of eastern Hokkaido, Japan in September-November 2021 was dominated by Karenia selliformis. C_LIO_LICells of Karenia selliformis typical in the bloom were discoid and possessing numerous small chloroplasts, approximately 70 in number. C_LIO_LICells of Karenia selliformis showed morphological variation in size and shape, and transparent motile cells lacking or having degraded chloroplasts were also present. C_LIO_LICo-occurred kareniaceans in the bloom were Karenia longicanalis, Karenia mikimotoi, Karlodinium sp. and Takayama spp. C_LIO_LIrDNA sequences of Karenia selliformis in the blooms of Hokkaido in 2021 and Kamchatka in 2020 were identical, which belong to the group I of Kr. selliformis. C_LI

ecology↗

bioRxiv2021

BET inhibitors synergize with anti-PD1 by rescuing TCF1+ progenitor exhausted CD8+ T cells in Acute Myeloid Leukemia

Many acute myeloid leukemia (AML) patients exhibit hallmarks of immune exhaustion, such as increased myeloid derived suppressor cells (MDSCs), suppressive regulatory T cells (Tregs) and dysfunctional T cells. We have developed a mouse model of AML driven by Flt3-ITD and Tet2 deficiency displays these immune-related features, including CD8+ T cells exhibiting a terminally exhausted phenotype (TEx). This T cell subset has been shown to be refractory to immune checkpoint blockade (ICB) monotherapy. Here we show that small molecule inhibitors which target bromodomain and extra-terminal domain (BET) proteins affect both tumor-intrinsic factors but also rescue T cell exhaustion and ICB resistance. Ex vivo treatment of cells from AML mice and AML patients with BET inhibitors (BETi) reversed CD8+ T cell exhaustion by restoring proliferative capacity and expansion of the more functional precursor exhausted T cells (TPEx). This reversal is enhanced by combined BETi and anti-PD1 treatment. Finally, we show that BETi synergizes with anti-PD1 in vivo, resulting in the reduction of circulating leukemia cells, enrichment of CD8+ T cells in the bone marrow, and increased expression of Tcf7, Slamf6, and Cxcr5 in CD8+ T cells. In total, we show the potential efficacy of combining BETi and ICB therapy in the treatment of AML.

cancer biology↗

bioRxiv2020

Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins

Ribosomes are the sophisticated machinery that is responsible for protein synthesis in a cell. Recently, quantitative mass spectrometry (qMS) based on data-dependent acquisition (DDA) have been widely used to understand the biogenesis and function of ribosomes. However, DDA-based qMS sometimes does not provide the reproducible and quantitatively reliable analysis that is needed for high-throughput hypothesis testing. To overcome this problem, we developed a highly sensitive, specific, and accurate method to quantify all ribosomal proteins (r-proteins) by combining selected reaction monitoring (SRM) and isotope labeling. We optimized the SRM methods using purified ribosomes and Escherichia coli lysates, and verified this approach as a high-throughput analytical tool by detecting 41 of the 54 r-proteins separately synthesized in E. coli S30 extracts. The SRM methods will enable us to utilize qMS as a high-throughput hypothesis testing tool in the research of E. coli ribosomes, and they have potential to accelerate the understanding of ribosome biogenesis, function, and the development of engineered ribosomes with additional functions.

synthetic biology↗