T lymphocyte activation plays a pivotal role in adaptive immune response and alters the spatial organization of nuclear architecture that subsequently impacts transcription activities. Here, using stochastic optical reconstruction microscopy (STORM), we observe dramatic de-condensation of chromatin and the disruption of nuclear envelope at a nanoscale resolution upon T lymphocyte activation. Super-resolution imaging reveals that such alterations in nuclear architecture are accompanied by the release of nuclear DNA into the cytoplasm, correlating with the degree of chromatin decompaction within the nucleus. The authors show that under the influence of metabolism, T lymphocyte activation de-condenses chromatin, disrupts the nuclear envelope, and releases DNA into the cytoplasm. Taken together, this result provides a direct, molecular-scale insight into the alteration in nuclear architecture. It suggests the release of nuclear DNA into the cytoplasm as a general consequence of chromatin decompaction after lymphocyte activation.