Background The role of T regulatory lymphocytes (Tregs) and their immunosuppressive mechanisms in the context of programmed death (PD)-1 blockade is not completely understood. Objectives To assess the impact of PD-1-blocking antibody treatment on Treg subpopulations in the blood. Methods We studied circulating Treg subpopulations in patients with melanoma under nivolumab or pembrolizumab treatment using flow cytometry and correlated these findings with clinical outcomes. Results These analyses revealed that the frequency of CD4 + CD25 ++ CD127 À PD-1 + lymphocytes (PD-1 + Tregs) significantly decreased after the first cycle of immunotherapy (23% vs. 8Á6%, P = 0Á043). Compared with patients who did not show a significant decline of PD-1 + Tregs after the first treatment, those who did had better clinical outcomes with respect to progression-free survival (PFS, P = 0Á022) and melanoma-specific death (MSD, P = 0Á0038). Multivariate analysis confirmed that a significant decline of PD-1 + Tregs in peripheral blood after the first treatment cycle is a significant predictor of more favourable PFS and MSD (P = 0Á04 and 0Á017, respectively). Interestingly, the occurrence of immune-related adverse events was also an independent predictor for decreased risk of MSD (P = 0Á047; odds ratio 0Á064, 95% confidence interval 0Á0042-0Á97). Conclusions We provide preliminary evidence that circulating PD-1 + Tregs rapidly decline after the initiation of treatment with PD-1-blocking antibodies, which is associated with reduced risk of melanoma progression and MSD. Patients showing no decrease of these PD-1 + Tregs in peripheral blood are characterized by an impaired response to immune checkpoint blockade and worse outcome.What's already known about this topic?• Programmed death (PD)-1-blocking antibodies are highly effective in melanoma treatment.• However, more than half of patients do not benefit from this therapy and to date it is difficult to predict which patients will respond to it.What does this study add?• PD-1-blocking antibody therapy rapidly results in a decline of circulating PD-1 + T regulatory cells (Tregs).

Upregulation of T-regulatory lymphocytes (Tregs) is one of numerous immune escape mechanisms of malignancies. In the present pilot study we aimed to study the effect of adjuvant nivolumab during the initiation of treatment on circulating Tregs subpopulations in patients with stage III melanoma. We subsequently recruited patients with stage III melanoma who had the indication for adjuvant anti-programmed death 1 (PD-1) treatment with nivolumab. Blood collections were performed before the initiation of nivolumab and before every 2-week therapy cycle. Flow cytometry was performed for the determination of circulating CD4+CD25highCD127−PD-1+(PD-1+Tregs) and CD4+CD25highCD127−CTLA-4+ (CTLA-4+Tregs) Treg populations. Circulating PD-1+Tregs [18.1% (range, 2.9%–41.7%) vs. 4.2% (0.4%–9.8%), P=0.0001] significantly decreased after the first cycle of immunotherapy and maintained decreased during a 3-month course of treatment. By contrast, CTLA-4+Tregs significantly increased after the first nivolumab dose when compared with CTLA-4+Tregs before the second treatment [0.75 (0–45.5) vs. 2.1 (0.1–90.8), P=0.0002]. Blood levels of PD-1+Tregs and CTLA-4+Tregs remained more or less decreased and increased during a 3-month therapy with nivolumab, respectively. Data of PD-1+Tregs as well as CTLA-4+Tregs was not significantly associated with frequencies of immune-related adverse events (P<0.05). In conclusion, we have demonstrated that circulating PD-1+Tregs of melanoma patients in stage III rapidly and continuously decline after the initiation of adjuvant treatment with the PD-1 blocking antibody nivolumab. By contrast, this decline is paralleled with an increase of CTLA-4+Tregs. The expression of PD-1 and CTLA-4 on Tregs might be a potential biomarker for the efficacy of immune checkpoint blockade in melanoma.

Background: Melanoma of unknown primary (MUP) is an uncommon clinical subtype of melanoma of known primary (MKP).Objectives: We aimed to compare treatment outcomes of MUP and MKP patients who had undergone therapy with immune checkpoint inhibitors (ICPI).Methods: We studied 41 metastatic melanoma patients (32 with MKP and 9 with MUP) with an indication for ICPI.Results: Clinical characteristics such as age, gender, stage of disease, etc., did not significantly differ (P < .05) between MUP and MKP patients. 20/32 (62.5%) melanoma-specific deaths (MSD) were observed in the MKP group, whereas 2/9 (22.2%) were detected in the MUP group (P = .035). On logistic regression, the MUP status proved to be an independent predictor for a more favorable outcome under immunotherapy when compared to MKP (P = .030).Conclusion: Our preliminary results indicate that MUP patients show better clinical outcome under ICPI when compared to MKP.