Data Augmentation for High-Fidelity Generation of CAR-T/NK Immunological Synapse Images

Chimeric antigen receptor (CAR)-T and NK cell immunotherapies have transformed cancer treatment, and recent studies suggest that the quality of the CAR-T/NK cell immunological synapse (IS) may serve as a functional biomarker for predicting therapeutic efficacy. Accurate detection and segmentation of CAR-T/NK IS structures using artificial neural networks (ANNs) can greatly increase the speed and reliability of IS quantification. However, a persistent challenge is the limited size of annotated microscopy datasets, which restricts the ability of ANNs to generalize. To address this challenge, we integrate two complementary data-augmentation frameworks. First, we employ Instance Aware Automatic Augmentation (IAAA), an automated, instance-preserving augmentation method that generates synthetic CAR-T/NK IS images and corresponding segmentation masks by applying optimized augmentation policies to original IS data. IAAA supports multiple imaging modalities (e.g., fluorescence and brightfield) and can be applied directly to CAR-T/NK IS images derived from patient samples. In parallel, we introduce a Semantic-Aware AI Augmentation (SAAA) pipeline that combines a diffusion-based mask generator with a Pix2Pix conditional image synthesizer. This second method enables the creation of diverse, anatomically realistic segmentation masks and produces high-fidelity CAR-T/NK IS images aligned with those masks, further expanding the training corpus beyond what IAAA alone can provide. Together, these augmentation strategies generate synthetic images whose visual and structural properties closely match real IS data, significantly improving CAR-T/NK IS detection and segmentation performance. By enhancing the robustness and accuracy of IS quantification, this work supports the development of more reliable imaging-based biomarkers for predicting patient response to CAR-T/NK immunotherapy.