Biology subjects

Tsunoda, M.

Publications and source records attributed to Tsunoda, M..

5 recordsLinked to original sources

bioRxiv2025

Autoclaving and long-term storage deplete glutamine sources in complex media and affect bacterial phenotypes

Autoclave sterilization is the most common method for sterilizing reagents and media in biology. However, the effects of heat-induced loss or modification of complex medium components on bacterial growth and phenotypes remain poorly understood. Here, we investigated the impact of autoclaving on glutamine sources in bacterial complex media using an Escherichia coli {Delta}glnA mutant, which requires exogenous glutamine for growth. {Delta}glnA exhibited impaired growth in LB medium after autoclaving compared with non-autoclaved LB, whereas its residual growth indicated the presence of heat-stable glutamine sources. Growth assays and HPLC quantification revealed that free glutamine in LB decreased from 58 {micro}M to 12 {micro}M upon autoclaving, while heat-stable glutamine sources remained at 117 {micro}M. Similar growth defects were observed for {Delta}glnA in autoclaved BHI, TSB, and M17 media compared with their non-autoclaved counterparts. Long-term storage of LB at room temperature for 24 weeks also reduced {Delta}glnA growth regardless of autoclaving, compared with freshly prepared LB. Furthermore, supplementation of glutamine sources into glutamine-deficient MHB medium enhanced biofilm formation by Pseudomonas aeruginosa. Collectively, these results demonstrate that autoclaving and storage reduce glutamine sources in complex media, thereby influencing bacterial growth and phenotypes.

microbiology↗

bioRxiv2024

LAG-3 blockade reactivates the CD8+ T cell expansion program to re-expand contracted clones in the tumor

Effective cancer immunotherapy relies on the clonal proliferation and expansion of CD8+ T cells in the tumor1,2. However, our insights into clonal expansions are limited, owing to an inability to track the same clones in tumors over time. Here, we developed a multi-tumor mouse model system to track hundreds of expanding and contracting CD8+ T cell clones over multiple timepoints in tumors of the same individual. Through coupling of clonal expansion dynamics and single-cell RNA/TCR-seq data, we identified a transcriptomic signature in PD-1+Ly108+ precursor exhausted cells3,4 that strongly predicts rates of intratumoral clone expansion in mice and humans. We found that expression of the signature successfully stratifies melanoma patient outcomes to PD-1/PD-L1 blockade5,6. Downregulation of the signature precedes clone contraction - a phase in which clones contract but maintain revivable precursor exhausted cells in the tumor. LAG-3 blockade - an FDA-approved therapy whose effects on CD8+ T cell responses are currently unclear7, re-activates the expansion signature, re-expanding pre-existing clones, including previously contracted clones. These findings reveal how the study of clonal expansion dynamics provide a powerful pan-immunotherapy signature for monitoring immunotherapies, including PD-1/PD-L1 and LAG-3 blockade, with implications for their future development.

immunology↗

bioRxiv2022

Clonal spreading of tumor-infiltrating T cells underlies the robust antitumor immune responses

The repertoire of tumor-infiltrating T cells is an emerging perspective for characterizing effective antitumor T-cell responses. Oligoclonal expansion of tumor T-cell repertoire has been evaluated; however, their association with antitumor effects is unclear. We demonstrated that the polyclonal fraction of the tumor-reactive T-cell repertoire consisting of relatively minor clones increased in tumor-bearing mice treated with anti-PD-L1 or anti-CD4 monoclonal antibody, which was correlated with antitumor effect. Meanwhile, the size of the oligoclonal fraction consisting of major clones remained unchanged. Moreover, the polyclonal fraction was enriched in progenitor exhausted T cells, which are essential for a durable antitumor response, and was more dependent on CCR7+ migratory dendritic cells, which are responsible for priming tumor-reactive T cells in the tumor-draining lymph nodes. These results suggest that the expansion of diverse tumor-reactive clones ("clonal spreading") is an important mechanism by which anti-PD-L1 and anti-CD4 treatments induce robust and durable antitumor T-cell responses.

immunology↗

bioRxiv2021

Role of TLR4 signaling on Porphyromonas gingivalis LPS-induced cardiac dysfunction in mice

Oral infections, particularly periodontitis, are a well-established risk factor for cardiovascular diseases, although the molecular mechanisms involved remain elusive. The aims of the present study were to investigate the effects of lipopolysaccharide derived from Porphyromonas gingivalis (PG-LPS) on cardiac function in mice, and to elucidate the underlying mechanisms. Mice (C57BL/6) were injected with PG-LPS (0.8 mg/kg/day) with or without an inhibitor of Toll-like receptor 4 (TLR4) signaling (TAK-242, 0.8 mg/kg/day) for 4 weeks. Left ventricular ejection function was significantly decreased at 1 week (from 67 {+/-} 0.5 to 58 {+/-} 1.2 %) and remained low at 4 weeks (57 {+/-} 1.0 %). The number of apoptotic myocytes was increased (approximately 7.4-fold), the area of fibrosis was increased (approximately 3.3-fold) and the number of 8-hydroxydeoxyguanosine-positive myocytes, a sensitive indicator of oxidative DNA damage, was increased (approximately 7.6-fold) at 4 weeks in the heart of PG-LPS treated mice. However, levels of various serum pro-inflammatory cytokines in PG-LPS-treated mice were similar to those in control mice. The impairment of cardiac function in PG-LPS-treated mice appears to involve activation of TLR4-NADPH oxidase (NOX) 4 signaling, leading to abundant production of reactive oxygen species and Ca2+ leakage from sarcoplastic reticulumn induced by calmodulin kinase II (CaMKII)-mediated phosphorylation of phospholamban (at Thr-17) and ryanodine receptor 2 (at Ser-2448). Pharmacological inhibition of TLR4 with TAK-242 attenuated the changes in cardiac function in PG-LPS-treated mice. Our results indicate that TLR4-NOX4 signaling may be a new therapeutic target for treatment of cardiovascular diseases in patients with periodontitis.

physiology↗

bioRxiv2021

T cell receptor (TCR) repertoire analysis reveals a highly conserved TCR repertoire in a bilateral tumor mouse model

Temporal analysis of the T cell receptor (TCR) repertoire has been used to monitor treatment-induced changes in antigen-specific T cells in patients with cancer. However, the lack of experimental models that allow temporal analysis of the TCR repertoire in the same individual in a homogeneous population limits the understanding of the causal relationship between changes in TCR repertoire and antitumor responses. A bilateral tumor model, where tumor cells were inoculated bilaterally into the backs of mice, could be used for temporal analysis of the TCR repertoire. This study examined the prerequisite for this strategy: the TCR repertoire is conserved between bilateral tumors with the same growth rate. Bilateral tumors with equivalent size and draining lymph nodes (dLNs) were collected 13 days after tumor inoculation to analyze the TCR repertoire of CD4+ and CD8+ T cells. The tumor-infiltrating T cell clones were highly conserved between the bilateral tumors, and the extent of clonal expansion was equivalent. In addition, the similarity between the bilateral tumors was equivalent to heterogeneity on one side of the tumor. The similarity of the TCR repertoire in the bilateral dLNs was markedly lower than that in the tumor, suggesting that tumor-reactive T cell clones induced independently in each dLN integrated during recirculation and then infiltrated the tumor. These findings suggest that our bilateral tumor model is suitable for temporal monitoring of the TCR repertoire to evaluate temporal and treatment-induced changes in tumor-reactive T cell clones. Significance StatementThe bilateral subcutaneous tumor model, where tumor cells were inoculated bilaterally into the backs of mice, is a promising model for temporal analysis of the antitumor response in cancer immunotherapy. This study demonstrated a highly conserved TCR repertoire in bilateral tumors and provided the basis for using a bilateral tumor model for evaluating temporal and treatment-induced changes in tumor-responsive T cell clones in individual mice. In humans, accurate statistical analysis is hampered by various background factors, including cancer type and stage and history of treatment. Moreover, temporal tumor biopsy in patients is highly invasive. Our bilateral tumor model overcomes these clinical issues and is expected to be a valuable tool for the development of novel immune monitoring and therapeutic strategies.

immunology↗