*2.2.1 Day-to-day profiling of T cell activation*

The assay was run using cryopreserved human PBMCs from a single healthy donor and were allowed to recover for 24 hours before use. On day 0, we stained human PBMCs with cell proliferation tracing dye (excitation: 488 nm and emission: 530/30 nm) and washed 3 times before plating into a 96-well plate at 2 million/mL (50 μL/well). We treated the cells by adding 50 μL/well of 3 different T-cell modulators in the respective wells: CD3/CD28 Dynabeads, PHA, or SEB. We performed an 11-point, 1:2 serial titration with duplicate wells per dose for each selected treatment. The final top concentration of the treatment in the assay well was 1 million/mL CD3/CD28 Dynabeads, 10 μg/mL PHA, or 100 ng/mL SEB, respectively. The untreated wells (with a concentration of zero) were used as negative controls for each treatment. On each day of culture (days 1, 3, and 6), we mixed the cell/supernatant sample in the culture plate by manual pipetting up and down 8 times. We transferred 10 μL cell/supernatant sample per well without dilution from the culture plate to a 96-well assay plate (Costar, Cat#3897), and stained the samples following the iQue® Human T Cell Activation Kit assay protocol. In the assay plate, 2 rows of wells (rows A and B) were designated for standard curve generation with a mixture of 2 protein standards (IFNγ and TNFα) in each well. Top concentration was 50,000 pg./mL for each protein in the standard mixture with 1:3 serial titration and duplicate wells per concentration. The lowest concentration was set to zero to determine background. Rows C-H were assigned to samples from 3 different treatment regimens: CD3/CD28 Dynabeads, PHA or SEB (**Supplemental Figure 1**). After staining and washing, samples from the full plate were acquired in approximately 15 minutes using the iQue® Screener PLUS (Sartorius), a high-throughput flow cytometry platform which has violet, blue and red lasers, and a total of 13 fluorescent channels. In each sample well of the assay plates, levels of secreted

cytokines (IFNγ and TNFα) were interpolated by reference to the corresponding standard curves generated from the standard wells in the same assay plate.

#### *2.2.2 Z' factor characterization of T cell activation*

The Z' factor of the assay was calculated to evaluate assay variation. The study was run similarly to the day-to-day profiling study described earlier, with minor modification. Cryopreserved human PBMCs from a single healthy donor were allowed to recover for 24 hours before use. On day 0 we plated the recovered PBMCs into a 96-well plate at 2 million/mL (50 μL/well). Then, we treated the cells by adding the same volume of CD3/CD28 Dynabeads. Total volume per well was 100 μL at the final concentration, with a total of 3 plates run. In each culture plate there were 24 wells without Dynabeads (negative control), and 24 treated wells with CD3/CD28 Dynabeads (positive control) with a final bead density of 1 million/ mL. After culturing 24, 48, or 72-hours, we mixed the cell/supernatant samples in the culture plate by manual pipetting 8 times. We transferred 10 μL of cell/supernatant sample per well without dilution, from the culture plates to 96-well assay plates and stained following the iQue® Human T Cell Activation Kit assay protocol. After staining and washing, samples from each plate were acquired on the iQue® Screener PLUS. iQue Forecyt® software was used to perform Z'-Factor plate calculations on well data of the positive and negative controls. For each plate, a single Z'- Factor was calculated as follows [Eq. 1]:

$$Z - factor = 1 - \frac{\mathfrak{Z}(\sigma\_p + \sigma\_n)}{\left| \mu\_p + \mu\_n \right|} \tag{1}$$


Positive control wells: CD3/CD28 Dynabeads-treated wells with bead/cell ratio at 1:1. Both Dynabeads and PBMC concentrations were at 1 million/mL. Negative control wells: cells only without CD3/CD28 Dynabeads.

#### **2.3 Kinase inhibitors screening**

#### *2.3.1 Culture setup for screening*

The cryopreserved PBMCs from a single healthy donor were allowed to recover for 24 hours before use. On day 0 we plated the recovered PBMCs into a 96-well plate at 4 million/mL (25 μL/well). We then added 25 μL of 40 μM kinase inhibitors (in culture media) into each well with cells to reach 20 μM inhibitor concentration. The negative control was culture media only and the positive control was 20 μM cyclosporine A. We mixed the culture and incubated for 1 hour with 5% CO2 at 37° C. We then added 50 μL (2 million/mL) of CD3/CD28 Dynabeads into each well of the screening culture plate. The final cell and Dynabead density were 1 million/mL each, and the final kinase inhibitor concentration was 10 μM. After mixing, the culture plate was incubated for 24 hours with 5% CO2 at 37°C.

*Developing a Novel Multiplexed Immune Assay Platform to Screen Kinase Modulators… DOI: http://dx.doi.org/10.5772/intechopen.97304*

#### *2.3.2 The screening assay*

The screening assay was performed after a 24-hour treatment. Because all wells of the 2 screening plates were filled with the kinase inhibitors and controls, we used a third plate for the standard curve generation. For the standard curve plate, only the top 2 rows were used. Top concentration for each standard in the mixture was 50,000 pg./mL, with a 1:3 serial titration and duplicate wells. The lowest concentration was zero for negative control purposes. After a 24-hour culture for the 2 screening culture plates, we mixed the cell/supernatant samples in the culture plates by manual pipetting up and down 8 times and then transferred 10 μL of the cell/ supernatant sample per well, without dilution from the 2 culture plates to the 2 screening assay plates. We stained the plates following the iQue® Human T Cell Activation Kit assay protocol. After staining and washing, the samples from each plate were acquired on the iQue® Screener PLUS. After data acquisition, the secreted levels of 2 cytokines (IFNγ and TNFα) in the 2 screening plates were interpolated using the iQue Forecyt® software by referring to the corresponding standard curves generated from the standard-only plate.

#### *2.3.3 Dosage test of kinase inhibitors*

After screening the kinase inhibitor library, we selected some compounds for further characterization by dosage testing. The test workflow was the same as the screening workflow described earlier with the exception of a series of kinase inhibitor concentrations. We performed 11-point, 1:2 serial titration with duplicate wells per dose for each selected compound. The final top concentration in the assay well was 10 μM for each selected compound. The concentration at zero μM was used as negative control (background) for each compound.

#### *2.3.4 General data acquisition and analysis method*

*The* iQue® Human T Cell Activation Kit comes with a template with predefined gates. Other analytical functions, such as heat maps, are queued to automatically populate. During data acquisition, events populated in the dot plots within the templated gates. The iQue QBeads® (for cytokine quantitation) and the cells were segregated based on size. Singlet beads were gated in a 2D plot of forward scatter (FSC)-height vs. FSC-area. After data acquisition on the iQue® Screener PLUS, all the cell and bead populations of interest were gated at the plate level. As shown in **Figure 1B**, the gating strategy separates cytokine capture beads from PBMC cells in an FSC vs. side scatter (SSC) plot, based on size and granularity differences. Singlet beads were gated in an FSC-height vs. FSC-area, and 2 different cytokine capture beads (iQue QBeads®) were separated in an RL1 (excitation: 640 nm; emission: 675/30 nm) vs. RL2 (excitation: 640 nm; emission: 780/60 nm) plot. Singlet cells are gated in an FSC-height vs. FSC-area plot. Live cells were gated in an RL1 vs. SSC plot. In live cell population, proliferated cells (dim fluorescent population) were gated from non-proliferated cells (bright fluorescent population) by using an overlay BL1 (excitation: 488 nm; emission: 530/30 nm) 1D histogram with positive and negative controls (data not shown). Also, from the live cell population, CD3+ T cells were separated from non-T cells in a CD3 vs. SSC plot. CD4+ T helper cells were separated from CD8 T cytotoxic cells in a CD4 vs. CD8 plot. CD69+, CD25+ and HLA-DR+ CD4 or CD8 T cells were gated in the corresponding plots with the markers at x-axis and SSC at y-axis. iQue Forecyt® software generated the standard curves (IFNγ and TNFα) using a 4-parameter logistic (4PL) regression fit with 1/Y<sup>2</sup>

#### **Figure 1.**

*Develop a multiplexed T cell activation assay. (A) a novel multiplexed T cell activation assay in a cell and bead mixture format. T cells in each assay well are stained with cell viability dye to differentiate live cells and dead cells. Cells are stained with CD3, CD4 and CD8 fluorescent antibodies to identify the CD4+ T helper cells and CD8+ T cytotoxic cells. Different T cell activation status is analyzed by staining with cell surface markers: CD69 (early activation marker), CD25 (late activation markers), and HLA-DR (even later activation markers). In the same assay well, the secreted IFNγ and TNFα after T cell activation are simultaneously measured by 2 cytokine capture beads in a sandwich immunoassay format. (B) the gating strategy for digital separation of the cytokine beads and the activated T cells acquired by high-throughput flow cytometry. After the sample from each well is acquired by iQue® screener PLUS, the cell/bead events are analyzed by iQue Forecyt® software (Sartorius), and cells and beads can be separated, based on the size and granularity, in FSC/SSC plot. IFNγ and TNFα capture beads are separated, based on the different bead intrinsic fluorescence in red fluorescence channels (RL1 and RL2 with excitation both at 640 nm, and with emission 675/30 nm and 780/ 60 nm, respectively). For cell phenotyping gating, live cells are separated from dead cells which are brightly stained by the cell viability dye intercalated with DNA. Live cells can be separated to CD3+ T cells and CD3 non-T cells. Based on CD4 and CD8 expression level, CD4+ T helper cells and CD8+ T cytotoxic cells are separated for further identification of early activation (CD69+), late activation (CD25+) and even later activation (HLA-DR+). (C) Representative data of time dependent profiling of 3 different modulators of T cell activation assay. 3 different modulators (CD3/CD28 Dynabeads, PHA and SEB) showed distinct signature of dose-dependent and time-dependent IFNγ secretion and the expression of early activation marker CD69. The unit is k/mL for CD3/CD28 Dynabeads, μg/mL for PHA, and ng/mL for SEB. Each data point represents mean* � *standard deviation (n = 2 wells).*

weighting factor. The linear range for each standard curve was generated automatically using iQue Forecyt® software with the following equations:

$$Y\_{Bend\ Lover} = \frac{(a-d)}{\frac{(1+1)}{k}} + d \tag{2}$$

$$Y\_{Bend\text{ }Bend\text{ }Higher\text{-}\frac{(a-d)}{(1+k)} + d\tag{3}$$

$$X\_{Bend} = c \left( \frac{a - Y\_{bend}}{Y\_{bend-d}} \right) \uparrow \frac{\mathbf{1}}{b} \tag{4}$$


*Developing a Novel Multiplexed Immune Assay Platform to Screen Kinase Modulators… DOI: http://dx.doi.org/10.5772/intechopen.97304*


Each cytokine concentration was interpolated by reference in the iQue Forecyt® software to the corresponding cytokine standard curve generated from the same assay plate, or from the standard-only plate.

#### *2.3.5 Algorithms used for hit identification*

We used a multi-plate analysis algorithm in the iQue Forecyt® software and a function called Profile Map based on Boolean logic to identify hits in the screening that simultaneously met multiple specified criteria across multiple plates. The hits were also ranked and compared in a line graph.

#### **3. Results**

#### **3.1 Assay characterization**

The goal of assay characterization was to evaluate assay robustness, verify the ability to differentiate the modulators of T cell activation, and determine if the assay would require optimization to screen a chemogenomic kinase library.

The assay was designed to achieve a wide dynamic range to detect high levels of these cytokines: IFNγ, linear range 91–22,204 pg./mL and TNFα, linear range 181– 50,000 pg./mL. The detection range is even wider than the linear range (data not shown). This wide dynamic range ensures the detection of high levels of secreted IFNγ and TNFα after T cell activation and eliminates a sample dilution step.

The assay variation was characterized and analyzed by measuring the Z' factor of 24-hour T cell activation with CD3/CD28 Dynabeads as a positive control and with untreated sample as a negative control. The mean Z' factor is 0.8 for both the percentage of CD69+ cells in CD4+ cells and for the percentage of CD69+ in CD8+ cells; 0.9 for both the percentage of CD25+ in CD4+ and for the percentage of CD25 + in CD8+ cells; 0.1 and 0.2 for the percentage of HLA-DR+ in CD4+ and CD8+ cells, respectively; 0.4 (IFNγ), and 0.7 (TNFα). As expected, a very low Z' factor for HLA-DR endpoint is a result of the late expression of this molecule after T cell activation. Forty-eight-hour and 72-hour activation did achieve higher HLA-DR Z' factor (0.3–0.5) than 24-hour activation. Different T cell activation timing may impact the signal of each endpoint and then, correspondingly, change the Z' factors. The Z' factors of 0.5 or higher in a multiplexed cell/bead-based mixture assay, suggest that the assay variation is appropriate for screening.

In order to evaluate the assay robustness in differentiating various compounds over different treatment time, we ran a day-to-day test monitoring of T cell activation. **Figure 1** shows the assay biochemistry (**Figure 1A**), and the cell/beads gating strategy (**Figure 1B**). **Figure 1C** of the day-to-day profiling results with 3 modulators demonstrated the assay robustness as a proof of concept. Only IFNγ and CD69+ cell endpoints are shown as examples. The results showed the 3 different

modulators had distinct profiles in IFNγ secretion, and in the expression of the early activation marker CD69. In addition, the results displayed a day-to-day effect. For the CD3/CD28 Dynabeads-treated condition, day 1 showed a dose-dependent IFNγ secretion, and day 3 and day 6 showed similar IFNγ secretion and saturation at a relatively high concentration. Even more interesting, the top 2 doses on day 3 and day 6 showed reduced IFNγ secretion ("hook" effect), which was consistent with the T cell exhaustion phenomenon characteristic of T cell activation [21]. Other endpoints also achieved distinct profiles including T cell proliferation determined by using a proliferation dye (data not shown).

### **3.2 General screening results**

As shown in **Supplement Figure 1**, screening the kinase inhibitor library (Cayman 152 kinase inhibitors) involved two assay screening plates.

Screening results of the kinase inhibitors on CD69 expression are shown in **Figure 2**, an iQue Forecyt® visualization function that displays a thumbnail dot plot of each well on the plate. This example shows a CD69 vs. SSC 2D plot. The figure shows only the first screening plate with CD4+ T cells as an example. Wells highlighted in bright red boxes, as examples in **Figure 2A**, show various inhibition of CD69 expression by inhibitors of 5 different kinase classes. **Figure 2B** shows a

#### **Figure 2.**

*The representative screening results: Kinase inhibitors decrease the expression of the early activation marker CD69 on CD4+ T helper cells in the plate 1. (A) the plate view of 2D plots (CD69 vs. SSC) that showed the CD69 expression in each well. Five screening wells with decreasing CD69 expression, as examples, were highlighted with red boxes. (B) the well-based 2D plots (CD69 vs. SSC) from five highlighted wells and a negative control well (A1 well) showed different decrease of early activation marker CD69 induced by different kinase inhibitors.*

*Developing a Novel Multiplexed Immune Assay Platform to Screen Kinase Modulators… DOI: http://dx.doi.org/10.5772/intechopen.97304*

zoomed-in view of each well highlighted in red and a negative control well (A1). The results suggest the assay can pick out different compounds as hits from different classes of kinase inhibitors.

To identify hits from the screen that meet multiple criteria, we used the iQue Forecyt® multi-plate and Profile Map algorithms to distinguish hits by dialing in the exact characteristics from multiple criteria. **Figure 3A** shows the 11 thresholds used to identify hits that decrease the cytokine secretion and the expression of these cell surface activation markers: IFNγ concentration, TNFα concentration, cell viability (Live Cells as % of total cells), CD4+ CD69+ as % of CD4+, CD8 + CD69+ as % of CD8+, CD4+ CD25+ as % of CD4, CD8+ CD25+ as % of CD8, CD4+ HLA-DR+ as % of CD4+, CD8+ HLA-DR+ as % of CD8+, Count of CD4+, Count of CD8+. HLA-DR was not critical in the criteria mix because it is a very late activation marker and is not highly expressed after 24 hours of activation. **Figure 3B** shows the wells (in blue) that meet the criteria specified in the iQue Forecyt® Profile Map. The positive control (cyclosporine A) is excluded from analysis (**Figure 3B**, column 12 on both plates) so that hits from only the samples can be generated for hit ranking (**Figure 3C**). The ranking is based on % CD69+ in CD4+ (from low to high). The IFNγ and TNFα level was normalized against the negative control as expressed at percentage (to fit into the same scale). A total of 25 hits showed broad inhibition of all major T cell activation markers including CD69, CD25, IFNγ, and TNFα. Interestingly, some patterns showed strong inhibition of cytokine secretion

#### **Figure 3.**

*The identification and ranking of the screening hits that inhibit broadly the secreted cytokine and the expression of the cell surface activation markers. Data were analyzed by iQue Forecyt® software's multi-plate analysis and profile map tools (Sartorius). (A) the 11 criteria used to identify the screening hits. All 11 criteria were applied simultaneously to help identify the screening hits. The criteria are subjective, based on these general concepts: Inhibition of T cell activation will decrease cytokine secretion and expression of cell surface activation markers; the "hit well" should have a pre-determined number of viable T cells; and, the desired percentage of hits. (B) the profile maps of two screening plates with a total of 25 screening hits (see blue boxes). The negative control, column 1 on both plates did not show hits as expected. Column 12 (positive controls) on both plates, which did show as positive (data not shown), were excluded from analysis for the hit ranking purpose. (C) the 25 screening hits out 152 kinase inhibitors from the whole library were ranked, based on the decrease (from low to high) of CD69 expression on CD4+ T cells (red curve) by using line graph feature in iQue Forecyt®. Other parameters were also shown for the 25 hits, including CD69 expression in CD8+ T cytotoxic cells, CD25 expression in CD4+ T helper and in CD8+ T cytotoxic cells, the normalized secretion of IFNγ and TNFα (normalized to the mean value of the negative wells, as expressed by the percentage).*

but medium inhibition of cell surface activation markers, while some compounds strongly inhibit almost every activation marker.
