Preprint Club
A cross-institutional Journal Club Initiative

Keywords

• Immunological synapse

• IL2 receptor distribution

• Supported-lipid bilayer model

Main Findings

T cell receptor (TCR) engagement during antigen presentation forms a stable contact between the T cell and antigen-presenting cell (APC), called the immunological synapse (IS). Multiple co-stimulatory receptors contribute to T cell activation within the IS, including the IL2 receptor (IL2R), which is expressed on T cells after antigen presentation. IL2R binds IL2 and signals primarily through the JAK/STAT pathway, with STAT5A and STAT5B being key activators. The IL2R consists of three subunits (IL2RA/B/G), which together form a high-affinity receptor. IL2 signaling is a target for immunotherapies, with high-dose IL2 already approved for treating metastatic melanoma and renal cell carcinoma. However, these therapies can cause toxic side effects by indiscriminately activating immune cells. A major challenge is improving the specificity of IL2-based therapies. One approach is linking IL2 signaling with TCR specificity. While there is evidence of crosstalk between IL2 and TCR signaling, their coordination within the IS remains unclear.

In this study, Capera et al. used a supported-lipid bilayer (SLB) system to mimic the IS and investigate IL2R distribution and activation during T cell engagement. They also used novel biologics, Immuno-STATs (ISTs), developed by Cue Biopharma. These ISTs selectively target and modulate disease-specific T cells by presenting tumor-specific peptides via MHC (pMHC) and IL2, aiming to activate T cells without triggering systemic immune responses.

Key findings from the study include the visualization of IL2R subunit distribution in naïve, memory, and activated CD8+ T cells, and the discovery of IL2R and IL2 in extracellular vesicles (EVs) released by activated T cells. These EVs induced pSTAT5 activation in naïve CD8+ T cells, suggesting a new mode of T-T cell collaboration. A spatiotemporal link between TCR and IL2R signaling was established, with pSTAT5 and pZAP70 overlaps observed. ISTs were found to simultaneously recruit and activate both TCR and IL2R subunits at the IS. When ISTs were present, there was a marked increase in pSTAT5 and pZAP70, along with the formation of homotypic synapses between T cells, indicating enhanced T-T cell collaboration or T-T cell crosslinking. These findings provide valuable insights into T cell signaling dynamics and suggest new strategies for enhancing T cell responses in immunotherapies, particularly through the use of IL2 Immuno-STATs.

Strengths of the preprint

The study provides important insights into the distribution of IL2R within the IS and the shedding of IL2R subunits in extracellular vesicles—both relatively unexplored areas that could inform improvements in IL2-based cancer therapies. The incorporation of novel immunotherapeutics not only validated the lipid bilayer model but also helped clarify the mechanism of action for these therapies. The research demonstrated that bispecific T cell engagers can selectively activate specific T cells, and that this activation can be enhanced by integrating IL2 into the therapeutics, offering potential strategies for more targeted and effective cancer treatments. As such, this preprint adds to a growing area of research using IL-2 bispecific-mediated co-stimulation to potentiate T cell responses.

Limitations & Suggestions

A limitation of the study is the reliance on confocal microscopy without incorporating other complementary methods to further support the claims and data. While a substantial number of cells were analysed, using additional techniques to demonstrate the correlation between IL2R and TCR expression or signalling, such as FACS, immunoblotting, or co-culture experiments, would have strengthened the findings. Though not critical, these methods could have made the conclusions more robust and convincing.

The study presents an intriguing possibility regarding IL-2 signaling in relation to EVs within the IS. However, the lack of in-depth questions, additional experiments, and follow-up discussions makes this aspect of the study feel insufficiently integrated. To enhance the novelty of the research, I suggest better incorporation of this topic or conducting further experiments to demonstrate the shedding of IL-2 receptors in EVs and its implications.

The study measured IL2R activity solely through pSTAT5 levels and TCR activity through pZAP70, even though ZAP70 is also active in non-activated T cells. Measuring additional downstream signals, such as Ca²⁺ signalling, would have provided stronger evidence for receptor activation. Including experiments with other effector readouts is crucial to fully validate their concept and make their findings more convincing. For example, the authors could consider measuring secretion of effector molecules into the supernatant when cells were treated with the biologicals.

The validation of the pharmaceuticals used in the study was solid, demonstrating that specific T cells can be activated by the biological. However, the specificity of this activation potential toxicity was not addressed. Since the authors included experiments with PBMCs, it would have been valuable to assess activation and toxicity in other T cell populations and cells more broadly. Testing for toxicity and off-target effects is crucial to make the study more biologically relevant and applicable to therapeutic settings.

Significance/Novelty

The development of bilayer models to simulate the IS is being explored by many labs, as is the creation of antibody-based therapeutics aimed at selectively activating T cells with IL2. While the combination of these two approaches was successful in this study, the novelty may not be entirely groundbreaking. However, given the therapeutic potential of IL2, enhancing our understanding of IL2R signalling remains crucial. Therefore, the relevance of this study is significant, as it could lead to important advancements in immunotherapeutic strategies and provides important pre-clinical data of the further clinical development of the biological itself.

Credit

Reviewed by Anna Schönbichler (Medical University Vienna) as part of a cross-institutional journal club between the Max-Delbrück Center Berlin, the Ragon Institute Boston (Mass General, MIT, Harvard), the Medical University of Vienna and other life science institutes in Vienna.

The author declares no conflict of interests in relation to their involvement in the review.