Given the complexity of the tumor microenvironment and the dynamic interaction between tumor and immune cells, PD-1/PD-L1/CTLA-4 regulatory pathways in tumors need a better understanding highlighting the need to investigate predictive biomarkers.
In malignant cells of various cancers, PD-L1 expression was graded from 0–50%. Malignant cells of melanoma were previously reported to express PD-L1 . However, we showed that malignant cell in melanoma lacked expression of PD-L1; this immune checkpoint protein was found only in antigen-presenting cells -macrophages (Fig. 2e,f, 3a) and dendritic cells (Fig. 3b, 4a,b). It means that PD-L1 expression by tumor cells cannot serve as an absolute biomarker of clinical response to checkpoint blockade in immunotherapy. This is in line with reports that patients, by which malignant cell in the tumor lack PD-L1 expression, also responded positively to PD-L1 checkpoint blockade therapies [29, 31, 32]. Correspondingly, patients with overexpressed PD-L1 in the tumor microenvironment, have improved clinical outcomes with anti-PD-1/PD-L1-directed therapy. It was earlier reported that PD-L1 is stronger presented in the tumor microenvironment then PD-1  and PD-L1 has been proposed as a potential target in cancer immunotherapy in human clinic [33, 35]. Therefore, PD-L1 expression in the tumor tissue can be regarded as a more valuable biomarker than PD-1 to guide clinical decisions.
The precise mechanism by which CTLA-4 regulates the immune response is complex and not fully understood. According to Pierre Golstein and colleagues, who were the first to identify CTLA-4[10, 37], CTLA-4 was originally defined as a T-lymphocyte antigen and later described as a T-cell surface receptor [11, 38–41], which, according to Allison's concept [12, 13], was later adopted as a target in melanoma immunotherapy. But we found that T-lymphocytes lacked the expression of CTLA-4, when using the anti-CTLA-4 antibody of the clone UMAB249, in human melanoma and in the tonsils also with the antibodies of AA 57–86 and SP355 clones (Fig. 1).
Most of the available data on CTLA-4 expression are limited to human T cells. Unfortunately, it is often ignored that CTLA-4 exists also in cells other than lymphocytes. When using the anti-CTLA-4 antibody of the clone UMAB249, we detected CTLA-4 in the melanoma in the tumor microenvironment cells, such as antigen-presenting cells ÷ macrophages (Fig. 2e,f, 3a) and dendritic cells (Fig. 3b, 4a,b), but not in T lymphocytes. In contrast, immunolabeling CTLA-4 with the antibody clone CAL49 vs T lymphocytes markers CD3, CD4, or CD8 showed that CTLA-4-positive T lymphocytes in human melanoma and tonsils were typically CD3 + and CD4+, but not CD8+ . Different immunolabeling pattern with anti-CTLA-4 antibodies of different clones can be explained by generating anti-CTLA-4 antibodies that recognize different epitopes of this protein. Further studies on this plausible controversy are warranted.
Despite the huge success and efficacy of the response to anti-CTLA-4 therapy in patients with melanoma, subsequent clinical trials have shown that combination therapy targeting both CTLA-4 and PD-1 appeared to be even more effective . This could be obviously explained by a massive T-lymphocyte presence in the tumor microenvironment observed in melanoma probes (Fig. 2b,d); most of CD3-positive cells co-expressed PD-1 (Fig. 5). Accordingly, better clinical outcomes with immune-checkpoint therapy in melanoma can also be awaited from combination therapy with the inclusion of PD-L1 as a target [44–46], while PD-L1 is strongly expressed in invaded macrophages in the melanoma tumor microenvironment (Fig. 2e,f) and in dendritic cells (Fig. 4a,b). This is in line with earlier reports that a high PD-L1 expression on immune cells, but not on tumor cells, is a favorable prognostic factor .