Cesium-137 and X-Ray Irradiation Yield Comparable Immune Phenotypes and Activation States in Bone Marrow Chimeric Studies

Bone marrow chimeras are widely used to study immune development and function. As the field moves from cesium-137 ( ¹³⁷ Cs)-based irradiators to X-ray irradiators for safety reasons, it is essential to determine if there are differences in immune system reconstitution after irradiating mice with one of these two radiation sources. Here, we performed a comprehensive immunological comparison of mice lethally irradiated with ¹³⁷ Cs or one of two different X-ray platforms and reconstituted with congenic bone marrow. Mice received 12 Gy total body radiation in two 6 Gy sessions followed by intravenous transfer of donor hematopoietic stem cells and were analyzed eight weeks post-transplant. We assessed mouse survival, donor chimerism, immune cell subset distribution, and activation states across multiple organs (bone marrow, spleen, lymph nodes, liver, and lung). All groups exhibited comparable survival and high levels of donor chimerism, with expected organ-specific reconstitution patterns. Immune lineage distributions, CD4/CD8 ratios, and activation states did not differ by irradiation type. Host-derived radioresistant cells were also similar across all irradiation groups and were predominantly composed of T cells skewed toward an activated phenotype. Overall, our data show that X-ray irradiation with proper filters and energy levels (225 KVp and 320 KVp) can yield equivalent immunological outcomes, including immune reconstitution and activation states, as compared to the same radiation dose from ¹³⁷ Cs-based irradiation in bone marrow chimera models. These results support the continued adoption of X-ray irradiation systems in place of ¹³⁷ Cs for generating bone marrow chimeras to be used across a wide range of immunologic studies.