Chimeric Antigen Receptor (CAR) T-cells represent a groundbreaking frontier in cancer treatment, offering a novel form of cellular immunotherapy. The recent FDA approvals for CAR T-cell therapy in B cell malignancies mark a significant milestone. With over 300 clinical trials underway globally, understanding the intricate cell biology of these “living” immunotherapies has become paramount. The rapid clinical translation of CAR T-cell therapy highlights the urgent need to delve deeper into how triggering T-cells through synthetic engineered receptors reshapes their biology, function, and long-term persistence. Previous research has demonstrated the remarkable ability of CAR T-cells to act as serial killers, a crucial attribute for effective anti-tumor responses and ultimately influencing the required dosage for successful therapy [1].
The activation of cytotoxic T-cells is initiated by the formation of an immune synapse, a highly organized structure at the interface of effector and target cells. This dynamic synapse is the hub of T-cell signaling, where serine kinases are recruited and key effector proteins like perforin and granzymes are secreted to induce target cell apoptosis. The synapse is characterized by concentric rings or SupraMolecular Activating Clusters (SMACs), often described as a “Bull’s eye” structure. T-cell receptor (TCR) signaling and termination occur in the central SMAC, while the peripheral SMAC provides adhesion, and actin clears away to the distal SMAC [2].
Recent investigations have focused on understanding how CAR-mediated T-cell triggering alters the immune synapse. Utilizing a dual-receptor transgenic mouse model expressing both the OTI TCR (specific for SIINFEKL peptide and H-2Kb) and a second-generation anti-Her2 CAR (CD28-CD3ζ) in the same T-cells [3], researchers compared immune synapse formation between TCR and CAR activation. The findings revealed that while TCR-mediated responses lead to the formation of a classical bull’s eye structure, CAR T-cell interactions exhibit a distinct pattern [4]. The CAR immune synapse is characterized by a disorganized multifocal signaling cluster, marked by Lck, which does not coalesce into a clearly defined structure. Notably, CAR T-cells do not form a defined peripheral SMAC, and unlike TCR-mediated interactions, they do not rely on LFA-1 interactions for synapse stabilization. Similar studies have reported a disorganized pattern of Zap70, a signaling molecule downstream of Lck, in CD19-specific CAR T-cells [5]. The strength of the signal received by the T-cell dictates its functional outcome. The influence of different CAR co-stimulatory domains and overall CAR design on synapse formation and downstream signaling remains an area of active investigation. However, the disorganized nature of the CAR synapse appears consistent across affinities and species, as CAR T-cells recognizing diverse antigens all display patchy signaling domains and actin clearance at the synapse [4].
Our current research highlights that CAR-mediated proximal signaling is faster compared to TCR signaling, suggesting potential for fine-tuning high-affinity CAR designs [4]. CAR T-cells also demonstrate a more rapid recruitment of lysosomes to the immune synapse, indicating a quicker killer response compared to TCR triggering. Signal strength is influenced by antigen binding affinity, interaction avidity, and synapse dwell time, resulting in a graded response to TCR signaling [6]. Previous studies have shown a correlation between T-cell-target synapse dwell time and cytokine and chemokine production [7], and CAR T-cells exhibit a similar off-rate compared to TCR interactions [1]. Interestingly, studies have indicated that lower affinity CAR T-cells can exhibit more efficient tumor clearance [8], making the relationship between CAR T-cell synapse off-rate, affinity, and function a key area of ongoing research.
In conclusion, our recent findings underscore the critical need for a deeper understanding of the mechanisms by which CAR T-cells eliminate target cells. This knowledge is crucial for enhancing the efficiency of this promising therapy. While CAR T-cells have proven highly effective in certain hematological cancers, their application to solid tumors presents additional challenges. As CAR T-cell therapy evolves, ongoing clinical investigations are exploring combinations with checkpoint inhibitors like anti-PD-1/CTLA-4 and other chemotherapeutic agents. Current CAR T-cell clinical trials utilize diverse CAR designs, and variations in affinity and design can lead to different signaling thresholds and functional outcomes. Ultimately, the functional capabilities of CAR T-cells are fundamentally influenced by their molecular design, emphasizing the importance of continued research and optimization in this rapidly advancing field of cancer immunotherapy.
Footnotes
CONFLICTS OF INTEREST
The authors declare no potential conflicts of interest.
REFERENCES
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