Biology subjects

Forsberg, M. H.

Publications and source records attributed to Forsberg, M. H..

3 recordsLinked to original sources

bioRxiv2024

Low Dose Radiation by Radiopharmaceutical Therapy Enhances GD2 TRAC-CAR T Cells Efficacy in Localized Neuroblastoma

BackgroundWhile chimeric antigen receptor (CAR) T cells have achieved significant success against hematological malignancies, efficacy against neuroblastoma has been limited. Virus-free CRISPR-edited GD2 TRAC-CAR T cells have been developed as a potential means of improving CAR T efficacy but are not curative. Radiopharmaceutical therapy (RPT) is a promising approach to enhance the effectiveness of immunotherapies, including immune checkpoint inhibitors. However, it remains unclear whether RPT can synergize with GD2 TRAC-CAR T cells to improve outcomes in neuroblastoma. MethodsDosimetry studies were conducted to measure the absorbed radiation dose delivered by lutetium-177 (177Lu) in both in vitro and in vivo models. Tumor-bearing mice were treated sequentially with low dose radiation by 177Lu-NM600, an alkylphosphocholine mimetic radiopharmaceutical agent, followed 9 days later by GD2 TRAC-CAR T cells generated in a virus-free manner by CRISPR/Cas9. Tumor burden was monitored through bioluminescence imaging and tumor size measurements. Mechanistic studies were performed using flow cytometry, multiplex assay and single-cell proteomic analysis. ResultsLow dose radiation delivered by 177Lu-NM600 synergized with GD2 TRAC-CAR T cells in a localized neuroblastoma model, resulting in complete tumor regression in all mice. The optimal combination was dependent on both the radiation dose and timing to minimize the negative impact of radiation on CAR T cell viability. Irradiation of neuroblastoma cells by low-dose RPT before GD2 TRAC-CAR T cells enhanced the release by CAR T cells of perforin, granzyme B and cytokines like TNF- and IL-7 while abrogating TGF-{beta}1 secretion. Additionally, low-dose RPT upregulated Fas on neuroblastoma cells, potentially enabling a CAR-independent killing. ConclusionsThis study demonstrates that low-dose RPT can enhance CAR T cell efficacy to treat a solid tumor. Findings suggest that optimization of radiation dose and timing may be needed for each patient and RPT to account for effects of varied tumor radiosensitivity and dosimetry. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/621668v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@153ff5dorg.highwire.dtl.DTLVardef@1a269b7org.highwire.dtl.DTLVardef@1ca9a53org.highwire.dtl.DTLVardef@59f461_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Metabolic priming of GD2 TRAC-CAR T cellsduring manufacturing promotes memory phenotypes while enhancing persistence

Manufacturing Chimeric Antigen Receptor (CAR) T cell therapies is complex, with limited understanding of how media composition impact T-cell phenotypes. CRISPR/Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant (TRAC) gene resulting in TRAC-CAR T cells with an enriched stem cell memory T-cell population, a process that could be further optimized through modifications to the media composition. In this study we generated anti-GD2 TRAC-CAR T cells using "metabolic priming" (MP), where the cells were activated in glucose/glutamine low media and then expanded in glucose/glutamine high media. T cell products were evaluated using spectral flow cytometry, metabolic assays, cytokine production, cytotoxicity assays in vitro and potency against human GD2+ xenograft neuroblastoma models in vivo. Compared to standard TRAC-CAR T cells, MP TRAC-CAR T cells showed less glycolysis, higher CCR7/CD62L expression, more bound NAD(P)H activity and reduced IFN-{gamma}, IL-2, IP-10, IL-1{beta}, IL-17, and TGF{beta} production at the end of manufacturing ex vivo, with increased central memory CAR T cells and better persistence observed in vivo. Metabolic priming with media during CAR T cell biomanufacturing can minimize glycolysis and enrich memory phenotypes ex vivo, which could lead to better responses against solid tumors in vivo.

bioengineering↗

bioRxiv2023

Autofluorescence lifetime imaging classifies human lymphocyte activation and subtype

New non-destructive tools are needed to reliably assess lymphocyte function for immune profiling and adoptive cell therapy. Optical metabolic imaging (OMI) is a label-free method that measures the autofluorescence intensity and lifetime of metabolic cofactors NAD(P)H and FAD to quantify metabolism at a single-cell level. Here, we investigate whether OMI can resolve metabolic changes between human quiescent versus IL4/CD40 activated B cells and IL12/IL15/IL18 activated memory-like NK cells. We found that quiescent B and NK cells were more oxidized compared to activated cells. Additionally, the NAD(P)H mean fluorescence lifetime decreased and the fraction of unbound NAD(P)H increased in the activated B and NK cells compared to quiescent cells. Machine learning classified B cells and NK cells according to activation state (CD69+) based on OMI parameters with up to 93.4% and 92.6% accuracy, respectively. Leveraging our previously published OMI data from activated and quiescent T cells, we found that the NAD(P)H mean fluorescence lifetime increased in NK cells compared to T cells, and further increased in B cells compared to NK cells. Random forest models based on OMI classified lymphocytes according to subtype (B, NK, T cell) with 97.8% accuracy, and according to activation state (quiescent or activated) and subtype (B, NK, T cell) with 90.0% accuracy. Our results show that autofluorescence lifetime imaging can accurately assess lymphocyte activation and subtype in a label-free, non-destructive manner. TeaserLabel-free optical imaging can assess the metabolic state of lymphocytes on a single-cell level in a touch-free system.

bioengineering↗