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Immunophenotypic characterization of human primary and metastatic melanoma infiltrating leukocytes
1Department of Pathology, School of Medicine, University of Southern California, Los Angeles 90033, USA.
During a systematic screening of melanoma infiltrating mononuclear and polynuclear immunological effector cells we employed 10 well characterized monoclonal antibodies, directed against leukocyte differentiation antigens, and the alkaline phosphatase conjugated, indirect, streptavidinbiotin immunocytochemical antigen detection technique and when necessary, the recently developed antigen retrieval method to enhance the intensity of the immunoreactivity to characterize the heterogeneous infiltrate of human primary (n = 30) and metastatic (n = 10) melanomas. Our study is the first to employ formalin fixed, paraffin embedded tissue sections in seeking to determine the ex vivo presence of tumor infiltrating leukocytes, including T lymphocytes, in human melanomas. We established the presence of some type of melanoma infiltrating host's immunological effector cells in all 40 observed melanoma cases'. More specifically, we found NK cells, macrophages and granulocytes in 30 out of 30 PMs and 10 out of 10 MMs. These effector cells represented the vast majority (> 80%) of the melanoma infiltrating immunocompetent cells. T lymphocytes were observed in 20 out of 30 PMs and 6 out of 10 MMs, but their numbers represented only between 5% to 10% of the heterogeneous leukocytic infiltrate. B cells were found in 22 out of 30 PMs and 8 out 10 MMs, their numbers representing less than 5%. Presence of cells of the dendritic reticulum, involved in antigen presentation was not determined in any of the observed PMs and MMs. There were more than enough macrophages and neutrophils, cells which are deeply involved in antigen presentation and, as previously mentioned, we found these cells within the melanomas. Thus defective antigen presentation cannot be the reason for the small proportion of T lymphocytes. What is to blame is probably the increased dedifferentiation of the melanoma immunophenotype (IP) to a degree that MHC class I molecules and the antigens complexed with them are lost and therefore there is nothing to be recognized by the T lymphocytes. In view of these results it is our-opinion that following the initial non-specific immune reaction, which may not be tumor specific, the specific immune response of effector T cells takes over. After significant IP changes by the majority of melanoma cells, including the internalization or shedding of MHC class I antigens, these highly specialized cells are no longer effective and the immune system responds by causing the reappearance of NK cells, macrophages and granulocytes. Thus, the relationship between melanoma and the host's immune system is evolutionarily dynamic: changes are readily made by either side when necessitated.
During a systematic screening of melanoma infiltrating mononuclear and polynuclear immunological effector cells we employed 10 well characterized monoclonal antibodies, directed against leukocyte differentiation antigens, and the alkaline phosphatase conjugated, indirect, streptavidinbiotin immunocytochemical antigen detection technique and when necessary, the recently developed antigen retrieval method to enhance the intensity of the immunoreactivity to characterize the heterogeneous infiltrate of human primary (n = 30) and metastatic (n = 10) melanomas. Our study is the first to employ formalin fixed, paraffin embedded tissue sections in seeking to determine the ex vivo presence of tumor infiltrating leukocytes, including T lymphocytes, in human melanomas. We established the presence of some type of melanoma infiltrating host's immunological effector cells in all 40 observed melanoma cases'. More specifically, we found NK cells, macrophages and granulocytes in 30 out of 30 PMs and 10 out of 10 MMs. These effector cells represented the vast majority (> 80%) of the melanoma infiltrating immunocompetent cells. T lymphocytes were observed in 20 out of 30 PMs and 6 out of 10 MMs, but their numbers represented only between 5% to 10% of the heterogeneous leukocytic infiltrate. B cells were found in 22 out of 30 PMs and 8 out 10 MMs, their numbers representing less than 5%. Presence of cells of the dendritic reticulum, involved in antigen presentation was not determined in any of the observed PMs and MMs. There were more than enough macrophages and neutrophils, cells which are deeply involved in antigen presentation and, as previously mentioned, we found these cells within the melanomas. Thus defective antigen presentation cannot be the reason for the small proportion of T lymphocytes. What is to blame is probably the increased dedifferentiation of the melanoma immunophenotype (IP) to a degree that MHC class I molecules and the antigens complexed with them are lost and therefore there is nothing to be recognized by the T lymphocytes. In view of these results it is our-opinion that following the initial non-specific immune reaction, which may not be tumor specific, the specific immune response of effector T cells takes over. After significant IP changes by the majority of melanoma cells, including the internalization or shedding of MHC class I antigens, these highly specialized cells are no longer effective and the immune system responds by causing the reappearance of NK cells, macrophages and granulocytes. Thus, the relationship between melanoma and the host's immune system is evolutionarily dynamic: changes are readily made by either side when necessitated.