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.

中文翻译：

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164P CD137和1型功能的组合检测可改善活化T淋巴细胞库的鉴定和表征

抽象的

背景

CD137（4-1BB）被广泛认为是T细胞活化的全面标志，它广泛用于癌症免疫学中以确定肿瘤反应性T细胞的比例。但是，CD137检测未提供有关激活的T细胞功能的特定信息。因此，在这项研究中，我们试图确定同时检测CD137以及常见的抗肿瘤免疫功能（1型功能，例如细胞因子TNFα和IFN-γ的产生，或CD107a的上调）是否可行，并提供了分别使用这两种方法的优势。

方法

在体外测试了用于检测细胞因子的常见蛋白质转运抑制剂，例如布雷菲德菌素A（BFA）或莫能菌素（MN），对CD137的表面和细胞内表达的影响。肿瘤浸润淋巴细胞（TILs）的激活是通过非特异性刺激（PMA和伊诺霉素）或天然存在的肿瘤抗原（自体肿瘤细胞）实现的。TIL激活定义为通过流式细胞术对CD137（CD137 ⁺ TILs）和/或TNFα，IFNγ和CD107a（类型1功能⁺ TILs）中至少一种染色的CD4 ⁺或CD8 ⁺ TILs的比例。

结果

我们的结果表明，MN对检测表面CD137的能力影响很小，而BFA不管采用何种刺激，其影响都大得多。尽管如此，当在BFA和MN均存在的情况下，将1型功能检测与CD137的细胞内染色相结合时，仍可以获得可靠的结果。使用这种方法，我们确定了三个肿瘤抗原特异性T细胞亚群，其特征在于CD137和1型功能或不具有1型功能的CD137的联合表达，反之亦然。与单独使用的两种方法中的任何一种相比，该组合方法可以鉴定出更大比例的肿瘤反应性T细胞。实际上，仅CD137就能识别出46％的肿瘤反应性CD8 + TIL，而86％的肿瘤反应性CD8 + TIL对1型功能呈阳性。

结论

结合检测CD137和常见的1型功能代表了一种更全面的方法，可识别和表征肿瘤抗原特异性活化T淋巴细胞的全部组成，而与所识别的抗原无关。

负责研究的法人实体

Herlev和Gentofte医院。

资金

丹麦癌症协会的研究经费A11806和A12535；研究基金R233-2016-3728和R286-2018-991伦贝克基金会; Herlev和Gentofte医院研究委员会的临床科学家补助金。

揭露

所有作者均声明没有利益冲突。