High Content Screening for Immune Modulators

Cancer immunotherapies that target T cells via the blockage of immune checkpoints are a promising approach for cancer treatment. However, the response rates vary between cancer patients. Studies have shown that while immunotherapies work well for some cancer patients, other patients fail to respond to the treatment (1). Since T cell activation requires physical contact with antigen presenting cells (APCs) through the formation of immunological synapse (IS) (2), a better understanding of T cell-tumor cell interactions will be critical for the development of next-generation immunotherapy.

When T cells encounter APCs, antigen recognition by CD3/T cell receptor (TCR) complexes trigger a cascade of intracellular signaling that leads to T cell activation. Activated T cells form symmetric interfaces with APCs through the establishment of IS. Kupfer et al. described the IS as a highly organized bull’s eye structure with a central TCR-major histocompatibility complex (MHC) interaction cluster. This cluster is surrounded by LFA-1-ICAM-1 adhesion and CD45 (3, 4). One can visualize the formation of IS through multiplex immunofluorescence (3) to study antigen recognition, adhesion, or co-stimulation/checkpoint receptors between the T cell and the APC. This approach can be adapted to use as a high throughput screening platform for immune modulator of T cells. Numerous studies have shown that immunological synapse can be used to predict effectiveness of chimeric antigen receptor (CAR) cells (5, 6). At Visikol, we believe multiplex high content imaging of IS is an ideal platform for immune modulator drug screening. Therefore, Visikol has established and now offers services for multiplex high content imaging of IS.

As an example, when we co-culture Jurkat cells (T cells) and superantigen-loaded Raji cells (APCs), we can detect IS formation via immunofluorescence (Figure 1) high content confocal imaging and image analysis. Through this approach, we can observe the reorganization of the actin cytoskeleton and adhesion molecules LFA-1 and ICAM-1. At the interface between Jurkat cells (T cells) Raji cells (APCs), there is the formation of a membrane bridge that can be visualized through phalloidin staining. Both phalloidin, LFA-1 and ICAM-1 staining can be detected when Jurkat-Raji cells were in close proximity. Visikol specializes in advanced cell culture assays and imaging and has demonstrated the utility of high content multiplex imaging of IS as a versatile screening platform for immune modulators.

Figure 1: IS formation between Jurkat-Raji cells. IS formed between Jurkat and antigen-loaded Raji cells when they were in close proximity after 45 mins co-culture. Phalloidin (red), ICAM-1 (green), LFA-1 (gray) and DAPI (blue).


  1. Carbone DP et al. First-Line Nivolumab in Stage IV or Recurrent Non-Small-Cell Lung Cancer. N Engl J Med. (2017);376 (25): 2415–2426.
  2. Dustin ML. T-cell activation through immunological synapses and kinapses. Immunol Rev. (2008); 221: 77–89.
  3. Monks, CR et al. Three-dimensional segregation of supramolecular activation clusters in T cells. (1998) 395, 82–86.
  4. Johnson KG et al. A supramolecular basis for CD45 tyrosine phosphatase regulation in sustained T cell activation. Proc Natl Acad Sci U S A (2000); 97:10138–10143.
  5. Xiong et al. Immunological Synapse Predicts Effectivenessof Chimeric Antigen Receptor Cells. Molecular Therapy (2018); 26, 4: 963-975.
  6. Liu et al. The Role of Immunological Synapse in Predicting the Efficacy of Chimeric Antigen Receptor (CAR) Immunotherapy Cell Communication and Signaling (2020); 18:134

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