The evolutionary conservation of non-core RAG regions suggests significant roles that might involve quantitative or qualitative alterations in RAG activity. Off-target V(D)J recombination contributes to lymphomagenesis and is exacerbated by RAG2’ C-terminus absence in Tp53^−/− mice thymic lymphomas. However, the genomic stability effects of non-core regions from both Rag1^c/c and Rag2^c/c in BCR-ABL1⁺ B-lymphoblastic leukemia (BCR-ABL1⁺ B-ALL), the characteristics, and mechanisms of non-core regions in suppressing off-target V(D)J recombination remain unclear. Here, we established three mouse models of BCR-ABL1⁺ B-ALL in mice expressing full-length RAG (Rag^f/f), core RAG1 (Rag1^c/c), and core RAG2 (Rag2^c/c). The Rag^c/c (Rag1^c/c and Rag2^c/c) leukemia cells exhibited greater malignant tumor characteristics compared to Rag^f/f cells. Additionally, Rag^c/c cells showed higher frequency of off-target V(D)J recombination and oncogenic mutations than Rag^f/f. We also revealed decreased RAG cleavage accuracy in Rag^c/c cells and a smaller recombinant size in Rag1^c/c cells, which could potentially exacerbate off-target V(D)J recombination in Rag^c/c cells. In conclusion, these findings indicate that the non-core RAG regions, particularly the non-core region of RAG1, play a significant role in preserving V(D)J recombination precision and genomic stability in BCR-ABL1⁺ B-ALL.

Cytokine polyfunctionality is a well-established concept in immune cells, especially T cells, and their ability to concurrently produce multiple cytokines has been associated with better immunological disease control and subsequent effectiveness during infection and disease. To date, only little is known about the secretion dynamics of those cells, masked by the widespread deployment of mainly time-integrated endpoint measurement techniques that do not easily differentiate between concurrent and sequential secretion. Here, we employed a single-cell microfluidic platform capable of resolving the secretion dynamics of individual PBMCs. To study the dynamics of poly-cytokine secretion, as well as the dynamics of concurrent and sequential polyfunctionality, we analyzed the response at different time points after ex vivo activation. First, we observed the simultaneous secretion of cytokines over the measurement time for most stimulants in a subpopulation of cells only. Second, polyfunctionality generally decreased with prolonged stimulation times and revealed no correlation with the concentration of secreted cytokines in response to stimulation. However, we observed a general trend towards higher cytokine secretion in polyfunctional cells, with their secretion dynamics being distinctly different from mono-cytokine-secreting cells. This study provided insights into the distinct secretion behavior of heterogenous cell populations after stimulation with well-described agents and such a system could provide a better understanding of various immune dynamics in therapy and disease.