Expression analysis of molecules targeting regulatory T-cells in patients with oral squamous cell carcinoma.

Abstract

e14508 Background: Immune checkpoint blockade is an important cancer treatment, but its therapeutic efficacy varies among patients. In the tumor microenvironment, PD-1 blockade reactivates PD-1+CD8+ T cells while simultaneously enhancing immunosuppression by PD-1+ regulatory T cells (Tregs); therefore, the balance between these populations has been proposed as a useful response biomarker. Because Tregs are also key therapeutic targets, multiple clinical trials using Treg depletion therapy have been conducted worldwide, but therapeutic efficacy has yet to be confirmed. In this context, we profiled the selectivity and expression intensity of representative Treg-depleting target candidates [CD25, T-lymphocyte antigen-4 (CTLA-4), C-C chemokine receptor 4 (CCR4), and CCR8] on effector Treg (eTreg), and investigated which molecules and combinations are more effective as therapeutic targets. Methods: A retrospective study was conducted on 24 patients with oral squamous cell carcinoma who underwent surgery. Peripheral blood lymphocytes (PBLs) were isolated from 24 patients and tumor infiltrated lymphocytes (TILs) from 24 patients respectively. eTreg fraction (CD4+CD45RA-FOXP3hi) was determined by flow cytometric analysis. eTreg frequency in each site and expression of CCR4, CCR8, CD25, CTLA-4 and PD-1 on the eTregs were analyzed. And PD-1 expression on CD8+ T cells was also assessed. The distribution of Tregs within the tissues was also analyzed using multi-fluorescence immunohistochemistry. Results: PD-1 expression on CD8+ T cells (mean [SD], %) was 7.9 [3.4] in PBL and 26.1 [9.2] in TIL (p < 0.001). PD-1 expression on eTregs was 1.7 [0.8] in PBL and 19.8 [9.1] in TIL (p < 0.001). The PD-1+CD8+ T cell/PD-1+eTreg ratio was 8.4 [14.6] in PBL and 1.8 [1.3] in TIL (p = 0.038), and this ratio varied across patients. CCR4 expression on eTregs was higher in PBL than in TIL (93.6 [6.6] vs 61.2 [14.0], p < 0.001), whereas CCR8 expression was higher in TIL than in PBL (61.3 [23.0] vs 23.6 [13.8], p < 0.001); accordingly, the CCR4/CCR8 balance on TIL eTregs was heterogeneous among patients. CD25 was highly expressed in both sites with slightly higher expression in TIL (PBL vs TIL: 80.5 [6.6] vs 85.8 [6.6], p = 0.008). CTLA-4 expression on eTregs was higher in TIL than in PBL (26.1 [9.5] vs 6.4 [3.2], p < 0.001). In the 5 cases of immunostaining, the PD-1+CD8+ T cell/PD-1+ Treg ratio was 11.7 [14.7] in Tumor area and 7.0 [8.9] in Stroma area (p = 0.52). Conclusions: These results suggest that for optimal Treg targeting strategies in oral cancer, it may be desirable to select different target molecules in the tumor and peripheral blood. Furthermore, because the expression of each molecule varies between patients, the optimal target molecule may differ for each patient.