Multiplexed Nanoplasmonic Temporal Profiling of T-Cell Response under Immunomodulatory Agent Exposure

Abstract

Immunomodulatory drugs-agents regulating the immune response-are commonly used for treating immune system disorders and minimizing graft versus host disease in persons receiving organ transplants. At the cellular level, immunosuppressant drugs are used to inhibit pro-inflammatory or tissue-damaging responses of cells. However, few studies have so far precisely characterized the cellular-level effect of immunomodulatory treatment. The primary challenge arises due to the rapid and transient nature of T-cell immune responses to such treatment. T-cell responses involve a highly interactive network of different types of cytokines, which makes precise monitoring of drug-modulated T-cell response difficult. Here, we present a nanoplasmonic biosensing approach to quantitatively characterize cytokine secretion behaviors of T cells with a fine time-resolution (every 10 min) that are altered by an immunosuppressive drug used in the treatment of T-cell-mediated diseases. With a microfluidic platform integrating antibody-conjugated gold nanorod (AuNR) arrays, the technique enables simultaneous multi-time-point measurements of pro-inflammatory (IL-2, IFN-γ, and TNF-α) and anti-inflammatory (IL-10) cytokines secreted by T cells. The integrated nanoplasmonic biosensors achieve precise measurements with low operating sample volume (1 μL), short assay time (∼30 min), heightened sensitivity (∼20-30 pg/mL), and negligible sensor crosstalk. Data obtained from the multicytokine secretion profiles with high practicality resulting from all of these sensing capabilities provide a comprehensive picture of the time-varying cellular functional state during pharmacologic immunosuppression. The capability to monitor cellular functional response demonstrated in this study has great potential to ultimately permit personalized immunomodulatory treatment.

Keywords: T cells; cytokines; immunomodulatory therapy; localized surface plasmon resonance (LSPR); multiplexed immunoassay; nanoplasmonic biosensing; tacrolimus.

Figure 1

(a) Assay process involving Jurkat T-cell stimulation and tacrolimus administration. Prepared Jurkat T cells were activated by PMA and Ionomycine and incubated for 2 h in a 6-well plate. This was followed by TAC administration and incubated for 1 h for cytokine secretion pathway alteration. During the first 2 h incubation period, cell-culture supernatant samples were collected every 60 min, and samples were collected every 10 min after dosing TAC to the cells. (b) T-cell intracellular cytokine secretion pathway and cellular-level effect of TAC. (c) Multiplexed cytokine detection using LSPR nanoplasmonic biosensor microarray chip. Collected samples were directly loaded into the chip through the top sample-loading PDMS channels. The bottom glass substrate, coated with patterned antibody-functionalized AuNR particles, was covered with sample loading channels. (d) Dark-field image of four parallel AuNR array patterns and SEM image of individual AuNR biosensors immobilized on glass. Nonuniform nanoparticles surfaces show their antibody-coated surfaces. (e) Principle of LSPR dark-field intensity imaging of LSPR nanoplasmonic biosensor microarrays. The surface binding of a targeted antigen at the sensing surface causes the sensor image intensity to increase as a result of both the spectral redshift and intrinsic intensity enhancement of the AuNR scattering light. Measuring the intensity change enables us to quantify the amount of the analyte in the sample.

Figure 2

(a) Mapping of intensity variations at LSPR microarray sensing spots for four different types of cytokines at different concentrations. (b) Standard curves of purified IL-2, IFN-γ, TNF-α, and IL-10 obtained from LSPR nanoplasmonic biosensor microarray chip. These curves were obtained from the intensity images in (a). Our device allows for triplicate measurements for each sample analysis with three sets of four parallel LSPR sensor stripe patterns integrated within the same detection microfluidic channel, which minimizes measurement error.

Figure 3

Temporal cytokine secretion profiles of Jurkat T cells for (a) IL-2, (b) IFN-γ, (c) TNF-α, and (d) IL-10 during two serial incubation periods: (1) 2 h after PMA and Ionomycin stimulation and (2) 1 h after TAC administration. The label of “Con” represents data from TAC-free control measurement in the second incubation period with the PMA/Inomycin stimulated cells. The labels of “T0.1”, “T1”, and “T10” represent data from the second incubation period after dosing TAC at the concentrations of 0.1, 1, and 10 ng/mL, respectively. The schematics in (e) and (d) show AP-1-mediated T-cell secretion pathways of IL-2 and IL-10, respectively.

Figure 4

Time-course cytokine secret rate variations of Jurkat T cells for (a) IL-2, (b) IFN-γ, (c) TNF-α, and (d) IL-10 during the 1 h incubation period after TAC administration. At the time point at t = 120 min is the point at which the TAC administration takes place. The labels of “Con”, “T0.1”, “T1”, and “T10” represent the same conditions as in Figure 3.