A cellular platform for the evaluation of immune checkpoint molecules

Jutz, Sabrina; Hennig, Annika; Paster, Wolfgang; Asrak, Ömer; Dijanovic, Dejana; Kellner, Florian; Pickl, Winfried F.; Huppa, Johannes B.; Leitner, Judith; Schmitz, Peter

doi:10.18632/oncotarget.17615

Cited by 50 publications

(72 citation statements)

References 54 publications

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“…Therefore, we compared the widely used Jurkat line E6.1 (JE6.1) and Jurkat 76 (J76) cells regarding the expression of accessory receptors implicated in T cell activation processes. Pronounced differences were seen for the activating receptors CD2 and CD226, which potently promote the activation of primary human T cell as well as T cell lines [ 20 – 23 ]. J76 cells were negative for CD226 and expressed low levels of CD2, whereas high levels of both molecules were detected on the JE6.1 subline ( Supplementary Figure 1B ).…”

Section: Resultsmentioning

confidence: 99%

A Jurkat 76 based triple parameter reporter system to evaluate TCR functions and adoptive T cell strategies

et al. 2018

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Adoptive T cell therapy using TCR transgenic autologous T cells has shown great potential for the treatment of tumor patients. Thorough characterization of genetically reprogrammed T cells is necessary to optimize treatment success. Here, we describe the generation of triple parameter reporter T cells based on the Jurkat 76 T cell line for the evaluation of TCR and chimeric antigen receptor functions as well as adoptive T cell strategies. This Jurkat subline is devoid of endogenous TCR alpha and TCR beta chains, thereby circumventing the problem of TCR miss-pairing and unexpected specificities. The resultant reporter cells allow simultaneous determination of the activity of the transcription factors NF-κB, NFAT and AP-1 that play key roles in T cell activation. Human TCRs directed against tumor and virus antigens were introduced and reporter responses were determined using tumor cell lines endogenously expressing the antigens of interest or via addition of antigenic peptides. Finally, we demonstrate that coexpression of adhesion molecules like CD2 and CD226 as well as CD28 chimeric receptors represents an effective strategy to augment the response of TCR-transgenic reporters to cells presenting cognate antigens.

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Section: Resultsmentioning

confidence: 99%

A Jurkat 76 based triple parameter reporter system to evaluate TCR functions and adoptive T cell strategies

et al. 2018

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“…The function of the ON-switch in a CAR was analyzed by determining the activation of the transcription factors NFAT and NFκB in reporter Jurkat cells. These reporter cells were generated by introducing an NFAT::eCFP reporter construct into a highly sensitive NF-κB::eGFP Jurkat cell line described previously ( 57 ). A highly sensitive cell line containing both reporters was established using a screening strategy described previously ( 58 ).…”

Section: Methodsmentioning

confidence: 99%

A conformation-specific ON-switch for controlling CAR T cells with an orally available drug

Zajc

Dobersberger

Schaffner

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Molecular ON-switches in which a chemical compound induces protein–protein interactions can allow cellular function to be controlled with small molecules. ON-switches based on clinically applicable compounds and human proteins would greatly facilitate their therapeutic use. Here, we developed an ON-switch system in which the human retinol binding protein 4 (hRBP4) of the lipocalin family interacts with engineered hRBP4 binders in a small molecule-dependent manner. Two different protein scaffolds were engineered to bind to hRBP4 when loaded with the orally available small molecule A1120. The crystal structure of an assembled ON-switch shows that the engineered binder specifically recognizes the conformational changes induced by A1120 in two loop regions of hRBP4. We demonstrate that this conformation-specific ON-switch is highly dependent on the presence of A1120, as demonstrated by an ∼500-fold increase in affinity upon addition of the small molecule drug. Furthermore, the ON-switch successfully regulated the activity of primary human CAR T cells in vitro. We anticipate that lipocalin-based ON-switches have the potential to be broadly applied for the safe pharmacological control of cellular therapeutics.

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“…In addition to “classical” effects like recruitment of enzymes that counteract TCR mediated downstream signaling processes ( 69 , 70 ), it was found that CTLA-4 mediates a reversal of the “stop-signal” initiated upon cognate T cell-APC interaction and thereby prevents efficient cytokine production and proliferation ( 71 ). Although these mechanisms contribute to T cell inhibition, there is increasing evidence that CTLA-4 exhibits inhibitory functions that are independent of its intracellular moiety ( 72 – 74 ). Therefore, one unique property of CTLA-4 is that “extrinsic effects,” specifically its capacity to interfere with CD28 costimulation, critically contribute to its function as an attenuator of T cell immunity.…”

Section: Cytotoxic T Lymphocyte Antigen-4 (Ctla-4)mentioning

confidence: 99%

“…T cell reporter systems based on the human T cell line Jurkat are powerful tools to assess mechanisms of co-inhibition and to test immune checkpoint inhibitors. Although such reductionist assay systems for evaluating antibodies against PD-1, CTLA-4, BTLA, and LAG-3 are commercially available and have been described in the literature ( 72 , 137 – 140 ), a validated test system for antibodies targeting TIM-3 has not yet been described to our knowledge. A recent report by Sabins and colleagues demonstrated that a TIM-3 antibody that was used in several studies to target human TIM-3 could function as an agonist and promoted CD8 T cell differentiation through activation of mTORC1 ( 141 ).…”

Section: T Cell Immunoglobulin and Mucin-domain Containing Protein-3 mentioning

confidence: 99%

Not All Immune Checkpoints Are Created Equal

Linhares

Leitner

Grabmeier‐Pfistershammer

et al. 2018

Front. Immunol.

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Antibodies that block T cell inhibition via the immune checkpoints CTLA-4 and PD-1 have revolutionized cancer therapy during the last 15 years. T cells express additional inhibitory surface receptors that are considered to have potential as targets in cancer immunotherapy. Antibodies against LAG-3 and TIM-3 are currently clinically tested to evaluate their effectiveness in patients suffering from advanced solid tumors or hematologic malignancies. In addition, blockade of the inhibitory BTLA receptors on human T cells may have potential to unleash T cells to effectively combat cancer cells. Much research on these immune checkpoints has focused on mouse models. The analysis of animals that lack individual inhibitory receptors has shed some light on the role of these molecules in regulating T cells, but also immune responses in general. There are current intensive efforts to gauge the efficacy of antibodies targeting these molecules called immune checkpoint inhibitors alone or in different combinations in preclinical models of cancer. Differences between mouse and human immunology warrant studies on human immune cells to appreciate the potential of individual pathways in enhancing T cell responses. Results from clinical studies are not only highlighting the great benefit of immune checkpoint inhibitors for treating cancer but also yield precious information on their role in regulating T cells and other cells of the immune system. However, despite the clinical relevance of CTLA-4 and PD-1 and the high potential of the emerging immune checkpoints, there are still substantial gaps in our understanding of the biology of these molecules, which might prevent the full realization of their therapeutic potential. This review addresses PD-1, CTLA-4, BTLA, LAG-3, and TIM-3, which are considered major inhibitory immune checkpoints expressed on T cells. It provides summaries of our current conception of the role of these molecules in regulating T cell responses, and discussions about major ambiguities and gaps in our knowledge. We emphasize that each of these molecules harbors unique properties that set it apart from the others. Their distinct functional profiles should be taken into account in therapeutic strategies that aim to exploit these pathways to enhance immune responses to combat cancer.

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A cellular platform for the evaluation of immune checkpoint molecules

Cited by 50 publications

References 54 publications

A Jurkat 76 based triple parameter reporter system to evaluate TCR functions and adoptive T cell strategies

A Jurkat 76 based triple parameter reporter system to evaluate TCR functions and adoptive T cell strategies

A conformation-specific ON-switch for controlling CAR T cells with an orally available drug

Not All Immune Checkpoints Are Created Equal

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