Loss of Nr4a1 Causes an Elevated Expression of Co-Inhibitory Receptor:Ligand Axes and Results in a Diminished T Cell-Mediated Lymphoma Cell Killing in Aggressive Lymphoma

Introduction: Aggressive non-Hodgkin lymphoma represent the most common type of lymphoid malignancies with a five-year survival rate of around 60%. Despite effective initial treatment, one-third of all patients will experience a relapse within several months to years after initial remission or become resistant to therapy. Because of these major treatment challenges, further research to discover novel therapeutic strategies is urgently required. In previous studies, we found that NR4A1 expression correlates with inferior survival in DLBCL patients and that ectopic expression of NR4A1 induces apoptosis in vitro and suppresses lymphoma growth in xenografts, suggesting tumor suppressive properties. Therefore, the aim of this study was to comprehensively study the function of Nr4a1 loss in lymphomagenesis.

Material and Methods:

The EµMyc lymphoma mouse model was intercrossed with the Nr4a1-/- mouse, resulting in a cohort of EµMyc Nr4a1+/+ (n=134), EµMyc Nr4a1+/- (n=59) and EµMyc Nr4a1-/- mice (n=84). The mice were monitored until the onset of overt disease. To identify which genes are regulated by Nr4a1, RNA-Seq was performed on RNA from lymphomas of the EµMyc Nr4a1+/+ and EµMyc Nr4a1-/- mice (n=5 each). Next, we transplanted lymphoma cells of EµMyc Nr4a1-/- and EµMyc Nr4a1+/+ mice into immune-competent C57BL/6 mice and immune-deficient Fox Chase SCID beige mice (n=20 per genotype). Furthermore, we determined the expression levels of immune checkpoint molecules in our human diffuse large B cell lymphoma (DLBCL) cohort and correlated them to NR4A1 expression (NR4A1-high (n=21) and NR4A1-low (n=17) human DLBCL samples). Finally, to investigate the immune-regulatory function of Nr4a1, we performed co-culture cytotoxicity assays using OVA_257-264 peptide-pulsed EµMyc Nr4a1+/+ and EµMyc Nr4a1-/- lymphoma cells and Ctla4^+/+ and Ctla4^-/- OVA targeting CD8⁺ T cells derived from OT-I mice and measured T cell-mediated lymphoma cell lysis via flow cytometry, respectively.

Results: In our EµMyc lymphoma mouse model we observed that the loss of Nr4a1 leads to accelerated lymphomagenesis in vivo (median survival = 92 days for EµMyc Nr4a1-/-, 101 days for EµMyc Nr4a1+/-, and 123 days for EµMyc Nr4a1+/+ mice, p<0.001), concomitant with increased expression of components of the Pd1-Pdl1-Pdl2- and Ctla4-Cd80-Cd86-axes (at least 5-fold, p<0.01,). Immuno-competent, but not immune-deficient mice, transplanted with Nr4a1-deficient lymphoma cells exhibited rapid lymphoma development, diminished survival (median survival = 24 days in EµMyc Nr4a1-/- - and 31 days for EµMyc Nr4a1+/+- setting, p=0.003), and upregulation of the immune checkpoint components like in the primary model (at least 5-fold, p<0.01). In our human DLBCL cohort, low NR4A1 expression correlated with a higher expression of immune checkpoint molecules, largely resembling our mouse data. Furthermore, immunohistochemical analysis of PD1, PDL2, and PDL2 revealed a significant higher percentage of PD1+ reactive immune cells (23.3% vs. 6.75%, p=0.002) and a significantly higher PDL1 (immunoreactivity score: 6.5 vs. 2.3, p=0.020) and PDL2 (immunoreactivity score: 26.2 vs. 18.5, p=0.064) protein expression. To unravel the impact of Nr4a1 on T cell-mediated lymphoma cell killing and on the regulation of the Ctla-4-Cd80-Cd86-axis of aggressive lymphomas, we performed co-culture cytotoxicity assays using OVA_257-264 peptide-pulsed EµMyc Nr4a1+/+ and EµMyc Nr4a1-/- lymphoma cells and Ctla4^+/+ and Ctla4^-/- OVA targeting CD8⁺ T cells from OT-I mice. In these experiments, we observed a significantly diminished lymphoma cell killing in the EµMyc Nr4a1-/- setting after 16 and 24 hours (after 16h: 44.8% vs. 22.3%, p=0.024 and after 24h: 71% vs. 36%, p=0.015), when we used Ctla4^+/+ OT-1 CD8⁺ T cells. Interestingly, when we used Ctla4^-/- OT-1 CD8⁺ T cells for the cytotoxicity assays, lymphoma cell killing was enhanced in the EµMyc Nr4a1-/- setting compared to that using Ctla4^+/+ OT-1 CD8⁺ T cells (34.4% vs. 13.2%, p=0.017).

Conclusion: Our data suggest that Nr4a1 plays a critical role in regulating the licensing of immune evasion in aggressive lymphomas by regulating the expression of co-inhibitory/-stimulatory molecules (ligands and their receptors). Thus, Nr4a1 might act as a promising target for restoring anti-lymphoma immune responses.

Novak:Bristol Myers Squibb: Research Funding. Greinix:Gilead, Novartis, Sanofi, Cellgene: Consultancy; Amgen, Gilead, Novartis, Sanofi, Takeda, Therakos: Speakers Bureau.