PRDM1 Deletion and STAT3 Mutations Cooperatively Promote CD8⁺ T-Cell and NK-Cell Growth in Vitro

PRDM1 loss and STAT3 activating mutations commonly co-occur in several non-Hodgkin lymphomas, including ALK negative Anaplastic Large Cell Lymphoma (ALCL) and NK-cell lymphoma (NKCL). PRDM1 is a tumor suppressor gene in NKCL, and also plays important roles in the homeostasis and differentiation of CD8⁺ T-cells. STAT3 encodes a critical transcription factor involved in specific cytokine-mediated JAK/STAT pathway activation in lymphocytes, controlling proliferation, apoptosis, and other cellular functions. However, it remains unclear whether these two abnormalities cooperate during lymphomagenesis. Here, we introduced PRDM1 deletion and STAT3 mutants into primary human T- or NK-cells isolated from PBMCs of healthy donors and performed functional studies on the genetically modified cells.

When either of the two common STAT3 mutants, STAT3^Y640F or STAT3^D661Y, was transduced into T- or NK-cells, the transduced cells showed short term growth advantage but could not persist, indicating that STAT3 activating mutations may provide stimulatory signals to T- and NK-cells initially, but inhibitory pathways could be activated that prevent long-term sustainable growth. When we knocked out PRDM1 first and then transduced the cells with the STAT3 mutants, the double modified T- or NK-cells showed sustained competitive growth advantage during in vitro culture. The effects were specific to STAT3 mutants as transduction of wild-type (WT) STAT3 or the STAT5B^N642H mutant did not improve cell growth. These findings suggest that PRDM1 loss and STAT3 mutations synergistically promote growth in primary T- and NK-cells.

We further showed that PRDM1-null, STAT3^Y640F-transduced NK-cells displayed better survival and growth compared to PRDM1-deleted or WT cells with feeder cell stimulation. More importantly, the double mutant NK-cells were more independent of feeder cell stimulation, and they were able to expand when supplemented with a single cytokine (IL-15 or IL-2). This feature may be relevant to lymphoma development in vivo where stimulatory cytokines may be limiting. In addition, these double mutant NK-cells had reduced expression of exhaustion markers Tim-3 and Tigit.

Similarly, genetically modified CD8⁺ T cells with PRDM1 knockout and STAT3-mutant were found to express much lower levels of PD-1, Tim-3, and Tigit, an important feature that may promote their growth in vivo where ligands of these co-inhibitory molecules may be expressed in the microenvironment. These double mutant CD8⁺ T cells exhibited greater stem memory (T_scm) phenotype, marked by CD45RA⁺, CD62L⁺, and CCR7⁺, suggesting the switch to a memory rather than an effector program.

Taken together, our in vitro experiments using primary human cells have provided evidences for the functional cooperation between PRDM1 loss and STAT3 activating mutation in promoting CD8⁺ T- and NK-cell growth. Some features shown by the double mutant cells may facilitate their survival and growth more significantly in vivo. We are currently investigating the adoptive transfer of modified mouse or human cells into littermates or NSG-Tg(huIL15) mice respectively to observe the in vivo development and evolution of the transferred cells. The detailed mechanistic interactions between these two genetic abnormalities in lymphoma development will be further investigated.

No relevant conflicts of interest to declare.