深入研究肿瘤浸润性T细胞免疫表型

AUTHOR:

David Draper, PhD | Associate Director, Scientific Development

DATE:

August 2019

流式细胞术使用大抗体面板的优点是检测更多的细胞亚群。当需要一个全面的数据集，但可用于分析的肿瘤材料有限时，这种能力尤其重要。此外，含有大量抗体的面板也可用于对少数亚群进行深入的免疫表型分析，或对单个亚群进行更深入的分析。在这个技术焦点中，我们通过展示使用新的扩展CompT生成的数据来展示后一种能力™ 面板，一个16色面板，对T细胞活化和分化进行流式分析，其水平高于Covance服务组合中的任何其他面板。

The Expanded CompT™ panel builds upon the CompT™ panel, our most popular standard panel to examine CD4⁺和CD8⁺T细胞。这个改进的小组增加了效应和记忆T细胞标记，加上四个额外的标记分析T细胞活化和衰竭。表1描述了扩展CompT的组件™ 小组，并使用未经治疗的小鼠MC38结肠癌，图1说明了其门控和分析策略。

正如所有Covance T细胞板,分析开始with dead cell exclusion and subsequent CD45⁺immune cell delineation to gate on CD3⁺T cells (not shown). Figure 1A displays the downstream endpoints of CD4⁺和CD8⁺T细胞分析在Compt™和扩展的Compt™面板之间共用。这些包括CD69和PD-1，其在T细胞活化时上调。它们的表达与已耗尽的T细胞表型相关。^[1]另一个共享端点是CD8⁺通过使用Ki-67表达作为替代标记物来提供T细胞增殖。最后，CD4⁺检测T细胞以量化辅助性T细胞和调节性T细胞（Tregs）。图1B和1C说明了用于扩展CompT的添加端点™ 面板，下面将进一步介绍。

Analysis of effector and memory CD8⁺T cell differentiation is shown in Figure 1B. Conversion of T cells to a memory phenotype is important for the development of lasting immunological response to rechallenge in the context of both infection and cancer pathogenesis. CD44 and CD62L analysis enables the delineation of T cells into four differentiation states. These include naïve or inactivated T cells, activated effector T cells (Teff), effector memory (Tem) and central memory (Tcm) subsets. Tem and Tcm subsets can circulate but have tendencies to reside in non-lymphoid and lymphoid tissues, respectively.^[2]Recent reports have demonstrated that both of these subsets have distinct roles in the anti-tumor response. A third resident memory (Trm) population has more recently been described as playing an important role in controlling tumor growth in a variety of models and can be delineated using CD103, among other markers.^[3]扩展组件™ 面板可以定制为包括Trm细胞的分析。

The Expanded CompT™ panel includes ICOS, LAG-3, TIM-3, and granzyme B analysis, which are four intensively investigated biomarkers for T cell functionality (Figure 1C). The analysis of these targets alone and in combination can provide insight into the anti-tumor potential of CD8+ T cells. Evidence supports a co-stimulatory and anti-tumor role for ICOS receptor signaling, thus making ICOS an attractive therapeutic target.[4] Granzyme B is often used as a biomarker for cytolytic activity and can correlate with CD8+ T cell anti-tumor responses. Conversely, PD-1, LAG-3 and TIM-3 are inhibitory receptors and while the expression of these three receptors has been linked to T cell exhaustion, a growing body of data suggests heterogeneity among sub-populations exists within the exhausted PD-1 expressing CD8+ T cells.[5] This heterogeneity correlates with the expression pattern of these inhibitory receptors. This profile can help define different sub-populations that have distinct potential to be re-invigorated to proliferate and/or lyse tumor cells.[6] Figure 2 illustrates how the Expanded CompT™ panel can quantify cells with double and triple positive expression for inhibitory receptors and provide insight into the heterogeneous PD-1 expressing T cell subset and its functionality. Numerous other T cell activation and inhibitory receptors have been described and implicated in influencing tumor immune responses; these include TIGIT, OX-40, CD137, CTLA-4, and others. Covance has experience analyzing many of these markers in离体tumor analysis. With minimal developmental efforts, the Expanded CompT™ can be customized to meet your unique pre-clinical needs.

Covance can configure custom panels with up to 18 colors, which creates options for the MI-Expanded CompT™ panel. In addition to substituting or adding different T cell activation/exhaustion markers as described in the previous section, NK/NKT cell analysis is a potentially valuable endpoint. This is enabled by the addition of CD49b/CD335 markers to the panel (Figure 3).

NK and NKT cells are an important source of IFNγ, have indirect effects on enhancing CD8+ T cell anti-tumor responses, and can directly lyse tumor cells by releasing cytolytic granules such as granzyme B.[7,8] Other options include IFNγ, TNFα, or other cytokine analyses for a more in depth profile of PD1+ and PD1– CD8+ T cells. Or add CD103 analysis to examine resident memory T cells for a deeper memory T cell profile in the tumor. Covance’s team has extensive experience developing custom flow cytometry services. To learn more about how the Expanded CompT™ panel can be incorporated into it into your preclinical research, contact the scientists at Covance.