**1. Introduction**

Unraveling the complex biochemistry of the immune system in search of innovative therapies is a new frontier of drug discovery with hundreds of autoimmune disease therapies and thousands of immuno-oncology therapies in the global development pipeline [1, 2].

Hallmarks of autoimmune disease pathogenesis are abnormal CD4 and CD8 T cell activation [3]. Genetic defects, mutations and other mechanisms resulting in increased T cell activity are involved in many autoimmune pathologies making

them attractive targets for the direct inhibition of T cell activation [4]. T helper cells —characterized by the expression of the surface molecule CD4—release cytokines that shape the immune response and pathology in autoimmune and inflammatory diseases [5].

Cancer immunotherapies are moving to the forefront of cancer treatment with a variety of regimens, such as immune checkpoint blockade therapies targeting T cells' regulatory pathways to enhance T cell activation and its anti-tumor immune responses. Focusing on immune checkpoint inhibitors is a disruptive change in immuno-oncology. Rather than directly attacking the tumor cell itself, the checkpoint inhibition strategy is removing inhibitory pathways by targeting the molecules involved in T cell regulation that block effective anti-tumor responses [6]. Another type of cancer immunotherapy consists of bispecific antibodies designed to redirect immune cells to tumor sites where they induce immune synapse formation, immune cell activation, cytokine secretion and proliferation leading to tumor lysis [7].

The versatile T cell weapon can also be deployed directly via adoptive T cell transfer (ACT). ACT involves infusion of in vitro expanded antigen-specific lymphocytes such as chimeric antigen receptor T (CAR-T) cells or tumor infiltrating lymphocytes (TILs) to cancer patients to mediate antitumor effects [8–10]. The T cell-mediated tumor cell killing is through tumor antigen recognition, robust T cell activation, proliferation and in vivo cytotoxicity. Different types of cancer immunotherapies can be combined or given sequentially to further enhance the magnitude of antitumor immune response over single agents [11].

In the adaptive immune response, the binding of the T cell receptor to peptides complexed with the major histocompatibility complex (MHC) on antigenpresenting cells, along with engagement of co-receptors such as CD4 or CD8 and co-stimulatory molecules such as CD28, triggers an intricate signal cascade. The key to T cell-mediated immune responses are the multiple intracellular biochemical events involving the complex interaction of cytosolic tyrosine kinases and serine– threonine kinases [12]. Kinases as drug targets have been investigated for decades in autoimmune disease, cancer, and degenerative diseases [13]. The kinase signaling pathway through Raf/MEK/ERK1/2 is a major regulator of cell proliferation and survival, and hyper-activation of this pathway is associated with human tumor malignancies [14]. Currently 37 kinase inhibitors have received FDA approval for treatment of malignancies such as breast and lung cancer. There are 150 kinasetargeted drugs in clinical trials, and kinase-specific inhibitors are in the preclinical stage of drug development [15]. In addition, combinations of kinase inhibitors are being explored for the treatment of cancer, and preclinical and clinical data demonstrate that therapeutic combinations enhance primary antitumor responses and delay the onset of resistance [16]. Kinase signaling also regulates the immune system and modulates the tumor immune microenvironment. Inhibition of kinase activity such as phosphoinositide 3-kinase (PI3K) promotes anti-tumor immunity through direct enhancement of CD8+ T cell activation and suppression of T regulatory cells [17]. The combination of kinase inhibition with cancer immunotherapy is another emerging research area as demonstrated in recent preclinical studies that showed the kinase inhibitor of P21 Activated Kinase 4 (PAK4) boosted PD-1 therapy in animal models [18].

Whether it is the characterization of immune phenotype or function, or the screening or profiling of biological or small molecule drug candidates, highthroughput flow cytometry and its ability to perform multi-parametric analyses of single cells or particles is playing an increasingly pivotal role in quantifying the myriad aspects of drug discovery [19, 20]. Here we developed a novel, multiplexed T cell activation assay platform based on high-throughput flow cytometry and associated advanced data analysis algorithms. As a proof-of concept, we screened a

kinase chemogenomic compound library and identified kinase inhibitors with various inhibition profiles of T cell activation, monitored by sampling a small aliquot from a mixture of cells and beads in a microtiter plate.
