Combined Detection of CD137 and Type 1 Functions Improves Identification and Characterization of the Activated T Lymphocyte Repertoire

Abstract

Abstract Background CD137 (4-1BB) is widely considered a comprehensive marker of T cell activation, and it is largely used in cancer immunology to determine the proportion of tumor-reactive T cells. However, CD137 detection does not provide specific information regarding the functions of activated T cells. Hence, in this study we tried to determine whether the simultaneous detection of CD137 plus common antitumor immune functions (type 1 functions such as production of cytokines TNFα and IFN-γ, or upregulation of CD107a) was feasible and provided advantages over using both methods separately. Methods The influence of common protein transport inhibitors used to allow cytokine detection, such as Brefeldin A (BFA) or Monensin (MN), on surface and intracellular expression of CD137 was tested in vitro. Activation of tumor infiltrating lymphocytes (TILs) was achieved with unspecific stimuli (PMA and Ionomycin) or naturally presented tumor-antigens (autologous tumor cells). TIL activation was defined as the proportion of CD4+ or CD8+ TILs staining for either CD137 (CD137+ TILs) and/or at least one of TNFα, IFNγ and CD107a (Type 1 function+ TILs) via flow cytometry. Results Our results showed that MN had minimal impact on the ability to detect surface CD137, whereas BFA had a much larger effect regardless of the stimulus employed. Nonetheless, robust results could be obtained when type 1 functions detection was combined with intracellular staining of CD137 in the presence of both BFA and MN. Using this method, we identified three tumor-antigen specific T cell subpopulations, characterized by combined expression of CD137 and type 1 functions or CD137 without type 1 functions and vice-versa. The combined method allowed the identification of a much larger proportion of tumor-reactive T cells, over either of the two methods used alone. Indeed, CD137 alone identified 46% of the total tumor-reactive CD8+ TILs, while 86% of the total tumor-reactive CD8+ TILs were positive for type 1 functions. Conclusion Combined detection of CD137 and common type 1 functions represents a more comprehensive method to identify and characterize the total repertoire of tumor-antigen specific, activated T lymphocytes, regardless of the antigen recognized. Legal entity responsible for the study Herlev and Gentofte Hospital. Funding Research grant A11806 and A12535, The Danish Cancer Society; Research grant R233-2016-3728 and R286-2018-991 Lundbeck Foundation; Clinician-Scientist Grant from the Herlev and Gentofte Hospital Research Council. Disclosure All authors have declared no conflicts of interest.