Spontaneous partial loss of the OT-I transgene

Pritchard, Gretchen Harms; Cross, Eric W.; Strobel, Marjorie C.; Jameson, Stephen C.; Kedl, Ross M.; Hogquist, Kristin A.; Hunter, Christopher A.

doi:10.1038/ni.3411

Cited by 8 publications

(5 citation statements)

References 10 publications

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“…Having demonstrated the feasibility of NP‐pHLIPs to escape from the lysosome and translocate to the cytoplasm, we proceeded to test whether these nanovaccines can enhance cross‐presentation of antigens by DCs to efficiently prime CD8 + T cells. Immature CD103 + BMDCs were first incubated with the different vaccines for 24 h. OVA‐specific CD8 + T cells harvested from OT‐I transgenic mice (termed OT‐I T cells) were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and subsequently cocultured with the nanovaccine‐treated DCs for 72 h. CFSE dilution was measured by flow cytometry to evaluate OT‐I T‐cell proliferation ( Figure A). The average number of cell divisions (termed as Mean cycles) was calculated and is depicted in Figure B.…”

Section: Resultsmentioning

confidence: 99%

Endolysosomal‐Escape Nanovaccines through Adjuvant‐Induced Tumor Antigen Assembly for Enhanced Effector CD8⁺ T Cell Activation

Qiu

Valente

Dölen

et al. 2018

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The activation of tumor-specific effector immune cells is key for successful immunotherapy and vaccination is a powerful strategy to induce such adaptive immune responses. However, the generation of effective anticancer vaccines is challenging. To overcome these challenges, a novel straight-forward strategy of adjuvant-induced tumor antigen assembly to generate nanovaccines with superior antigen/adjuvant loading efficiency is developed. To protect nanovaccines in circulation and to introduce additional functionalities, a biocompatible polyphenol coating is installed. The resulting functionalizable nanovaccines are equipped with a pH (low) insertion peptide (pHLIP) to facilitate endolysosomal escape and to promote cytoplasmic localization, with the aim to enhance cross-presentation of the antigen by dendritic cells to effectively activate CD8 T cell. The results demonstrate that pHLIP-functionalized model nanovaccine can induce endolysosomal escape and enhance CD8 T cell activation both in vitro and in vivo. Furthermore, based on the adjuvant-induced antigen assembly, nanovaccines of the clinically relevant tumor-associated antigen NY-ESO-1 are generated and show excellent capacity to elicit NY-ESO-1-specific CD8 T cell activation, demonstrating a high potential of this functionalizable nanovaccine formulation strategy for clinical applications.

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Section: Resultsmentioning

confidence: 99%

Endolysosomal‐Escape Nanovaccines through Adjuvant‐Induced Tumor Antigen Assembly for Enhanced Effector CD8⁺ T Cell Activation

Qiu

Valente

Dölen

et al. 2018

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“…Murine B16F10 melanoma cells were chosen as a model due to their resistance to immune checkpoint blockade against receptors such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) 30,31 . OVA protein transduction was performed due to availability of transgenic mice with OT-I T cells that recognize OVA peptide of amino acids 257–264 (SIINFEKL) expressed in the context of H-2Kb 32,33 , which allows for antigen-specific T cell killing assays. Lentiviral infection occurred at a low multiplicity of infection (MOI) (MOI < 0.2) to bias towards single lentiviral sgRNA construct integration.…”

Section: Resultsmentioning

confidence: 99%

Double knockout CRISPR screen in cancer resistance to T cell cytotoxicity

Park

Codina

et al. 2022

Preprint

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Immunotherapy has transformed cancer treatments; however, a large fraction of patients encounter resistance. Such resistance is mediated by complex factors, often involving interactions between multiple genes. Thus, it is crucially important to identify genetic interactions between genes that are significantly mutated in cancer patients and those involved in immune responses, ideally the ones that currently have chemical compounds for direct targeting. To systematically interrogate such genetic interactions that mediate cancer cells response to T cell killing, we designed an asymmetric CRISPR Cas9 dual perturbation library targeting the matched combinations between significantly mutated tumor suppressors and immune resistance genes. We performed a combinatorial double knockout screen on 1,159 gene pairs and identified those where joint loss-of-function renders altered cellular response to T cell cytotoxicity. With individual double knockout constructs we validated these genetic interactions including Jak1-Trp53, Jak1-Kmt2d, and Ifngr1-Kmt2d. Interactions between significantly mutated tumor suppressors and potentially druggable immune resistance genes may offer insights on potential new concepts of how to target clinically relevant cancer mutations with currently available agents. This study also provides a technology platform and an asymmetric CRISPR double knockout library for interrogating genetic interactions between cancer mutations and immune resistance pathways under various settings.

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“…Nonetheless, the distribution of CD8 + T cells carrying the OVA 257-264 -specific TCR in OT-I +/− mice and the effect of aging on the maintenance of transgenic TCR expression have not been investigated so far. Besides, spontaneous partial loss of the V α 2 or V β 5 chain in OT-I +/+ mice has been reported in some cases (Pritchard et al, 2016).…”

Section: Preserved Expression Of Transgenic T-cell Receptor In Elderl...mentioning

confidence: 99%

Heterozygous OT‐I mice reveal that antigen‐specific CD8⁺ T cells shift from apoptotic to necrotic killers in the elderly

et al. 2023

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Numerous alterations in CD8 + T cells contribute to impaired immune responses in elderly individuals. However, the discrimination between cell-intrinsic dysfunctions and microenvironmental changes is challenging. TCR transgenic OT-I mice are utilized to investigate CD8 + T-cell immunity, but their immunodeficient phenotype hampers their use especially in aging. Here, we demonstrate that using a heterozygous OT-I model minimizes the current limitations and provides a valuable tool to assess antigen-specific T-cell responses even at old age. We analyzed phenotypic and functional characteristics of CD8 + T cells from OT-I +/+ and OT-I +/− mice to prove the applicability of the heterozygous system. Our data reveal that OVA-activated CD8 + T cells from adult OT-I +/− mice proliferate, differentiate, and exert cytolytic activity equally to their homozygous counterparts. Moreover, common age-related alterations in CD8 + T cells, including naive T-cell deterioration and decreased proliferative capacity, also occur in elderly OT-I +/− mice, indicating the wide range of applications for in vivo and in vitro aging studies. We used the OT-I +/− model to investigate cell-intrinsic alterations affecting the cytotoxic behavior of aged CD8 + T cells after antigen-specific in vitro activation. Time-resolved analysis of antigen-directed target cell lysis confirmed previous observations that the cytotoxic capacity of CD8 + T cells increases with age.Surprisingly, detailed single cell analysis revealed that transcriptional upregulation of perforin in aged CD8 + T cells shifts the mode of target cell death from granzymemediated apoptosis to rapid induction of necrosis. This unexpected capability might be beneficial or detrimental for the aging host and requires detailed evaluation.

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Spontaneous partial loss of the OT-I transgene

Cited by 8 publications

References 10 publications

Endolysosomal‐Escape Nanovaccines through Adjuvant‐Induced Tumor Antigen Assembly for Enhanced Effector CD8⁺ T Cell Activation

Endolysosomal‐Escape Nanovaccines through Adjuvant‐Induced Tumor Antigen Assembly for Enhanced Effector CD8⁺ T Cell Activation

Double knockout CRISPR screen in cancer resistance to T cell cytotoxicity

Heterozygous OT‐I mice reveal that antigen‐specific CD8⁺ T cells shift from apoptotic to necrotic killers in the elderly

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Spontaneous partial loss of the OT-I transgene

Cited by 8 publications

References 10 publications

Endolysosomal‐Escape Nanovaccines through Adjuvant‐Induced Tumor Antigen Assembly for Enhanced Effector CD8+ T Cell Activation

Endolysosomal‐Escape Nanovaccines through Adjuvant‐Induced Tumor Antigen Assembly for Enhanced Effector CD8+ T Cell Activation

Double knockout CRISPR screen in cancer resistance to T cell cytotoxicity

Heterozygous OT‐I mice reveal that antigen‐specific CD8+ T cells shift from apoptotic to necrotic killers in the elderly

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Endolysosomal‐Escape Nanovaccines through Adjuvant‐Induced Tumor Antigen Assembly for Enhanced Effector CD8⁺ T Cell Activation

Endolysosomal‐Escape Nanovaccines through Adjuvant‐Induced Tumor Antigen Assembly for Enhanced Effector CD8⁺ T Cell Activation

Heterozygous OT‐I mice reveal that antigen‐specific CD8⁺ T cells shift from apoptotic to necrotic killers in the elderly