T Cell CD62L Expression Following Nivolumab Therapy Is Associated with Long Term Response to Rituximab-Nivolumab in Treatment Naïve Follicular Lymphoma: Results from the 1st FLOR Study

Inter-tumoral T cell dysfunction and efficacy of immune checkpoint inhibitors (ICI) in follicular lymphoma (FL) has been well described. However, few studies have examined peripheral blood immunity at diagnosis or the impact of frontline ICI on circulating immunity in FL. We hypothesised that immune dysregulation in peripheral blood would be present in treatment naïve FL and would correlate to response to frontline ICI. PBMC samples from 34 untreated FL patients receiving rituximab (R) and nivolumab in the 1st FLOR trial (Hawkes JCO 2021) were collected at baseline, after 4 cycles of nivolumab (PET-CT2) and after 6 months of nivolumab±R (PET-CT4) as part of a pre-planned translational analysis. Immune profile was assessed using a single-tube 29 antibody panel on an Aurora spectral flow cytometer and compared to the immune profile of 12 age matched healthy donors. Results were correlated with centrally determined PET response (Lugano criteria). Compared to healthy donors, at baseline FL patients had increased proportions of TRegs (P<0.0001) with decreased HLA-DR+ (P<0.01) and increased PD-1+ (P<0.01) populations. NK cells were increased (P<0.05) with a 2-fold increase in TIM3 expression (P<0.01). While total T cells were unchanged, expression of PD-1, TIM3, HLA-DR and 4-1BB were significantly increased in both CD4 and CD8 T cells from FL patients. B cell and monocyte populations were unchanged. To investigate if peripheral blood immunity is related to tumour activity, total metabolic tumour volume (TMTV) was calculated. There were strong correlations between TMTV and circulating CD4+PD1+ T cells (P<0.01), Total TRegs (P<0.01) and TRegs expressing TIM3 (P<0.05) and PD1 (P<0.01) indicating the known tumour microenvironmental immune changes in FL are also reflected in circulating immunity and correlate with disease activity. The impact of circulating immunity on disease prognosis was determined by examining correlation with the FL International Prognostic Index (FLIPI). Patients with high risk disease (FLIPI3+) exhibited increased dysregulation of immune checkpoints across TRegs, CD4 and CD8 T cells at baseline. Treatment on study with nivolumab significantly increased the populations of TRegs expressing 41BB, LAG-3, TIM3 or PD-L2 (P<0.05) with a decreased PD-L1+ population at PET-CT2. Expression of immune checkpoints on T cells was unchanged by therapy and remained high compared to healthy donors. Biomarkers of disease relapse were assessed by exploring correlation with progression of disease within 24 months (POD24). 10 of 34 patients experienced POD24, which did not correlate with baseline TReg, CD4, CD8 or NK cell populations. At PET-CT2, there was reduced proportions of HLA-DR+ TRegs in patients that did not have POD24. To assess biomarkers of sustained response, patients were stratified into sustained CR (CR achieved by PET-CT4 and maintained for 6 months without evidence of relapse, n=15) vs PR/PD (n=21) and changes in immune profile assessed. At baseline, sustained CR was associated with a trend for increased proportions of Naïve CD4 and CD8 T cells and decreased TRegs and PD-L2 expressing TRegs. Baseline proportions of PD-1 expressing T and NK cells did not correlate with sustained response. Most strikingly, expression of CD62L was significantly downregulated across total CD4 and CD8 T cells and memory subsets and TRegs at PET-CT2 in PR/PD patients (P<0.05) and maintained at baseline levels in those patients who eventually achieved a sustained CR. At this early timepoint final patient responses had not been established with most patients having either progressive or stable disease, suggesting that dysfunctional downregulation of CD62L in response to nivolumab may reflect the inability of patient's T cells to activate and/or migrate to the tumour site and facilitate tumour clearance and long-term treatment responses. Overall, grade 1-3A treatment naïve FL was associated with significant dysregulation of peripheral blood T and NK cells prior to therapy including increased expression of multiple immune checkpoints. Early downregulation of CD62L on T cells of patients treated with nivolumab was associated with the inability to achieve long term complete responses and may indicate aberrant T cell activation and/or function which impacts on long term response to therapy and may be used to identify a population that requires additional early intervention.