Poster T4 - Using RT-qPCR as an Early Screening Method for Antigen Specific CD4 T Cell Activation

Saturday, October 26, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Maya Zhang, MSE (she/her/hers)

PhD Student
Georgia Institute of Technology
Atlanta, Georgia, United States

Co-Author(s)

Si-Sim Kang, MA (she/her/hers)

PhD Candidate
Johns Hopkins University
Baltimore, Maryland, United States
DT

Daniela Trejo Zambrano, PhD (she/her/hers)

PhD Student
Johns Hopkins School of Medicine
Timonium, Maryland, United States

Primary Investigator(s)

Jonathan P. Schneck, MD, PhD (he/him/his)

Professor
Johns Hopkins University, United States

Introduction: Antigen-specific T cells are crucial for immune responses against tumors. Detecting and quantifying these cells is essential for understanding immune responses and developing effective therapies. This study explores RT-qPCR as a rapid and sensitive method to evaluate patients' T cell responses to specific peptide-MHC complexes. This approach could significantly affect adoptive cell transfer (ACT) therapies targeting neo-antigens. By measuring the expression of critical cytokines like IFN-γ and IL-2, we aim to provide a fast and reliable technique for assessing antigen-specific T cell populations and their activation status.

Materials and

Methods: Unloaded artificial antigen presenting cells (aAPCs) were synthesized using magnetic nanoparticles conjugated with HLA-DP, with CLIP as a place holder, and α CD28. To load the aAPCs with target peptides, thrombin cleavage was used to exchange the placeholder CLIP molecule with DP4 restricted peptide p30 of tetanus toxoid (947-967) or HSV (283–302).
Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood from 6 donors using density gradient centrifugation and cryopreserved in FBS containing 10% DMSO. Freshly thawed PBMCs were cultured at 37°C in human T cell media with loaded or unloaded aAPCs for 3 hours or overnight. RNA was extracted following the Zymol Direct-Zol RNA Miniprep extraction protocol and stored at -80°C until use.
The 1-step RT-PCR reaction was performed using TaqMan Fast Virus 1-Step Master Mix. Quantitative real-time PCR was performed using QuantStudio 6 and QuantStudio Real-time PCR software. IFNG and IL2 Ct values were normalized to CD4 and HPRT1 mRNA for all assay conditions, and final data was reported as the relative fold change above the unstimulated controls using the comparative Ct (ΔΔCt) method.

Results, Conclusions, and Discussions:

Results:
After the initial 3- hour incubation with aAPCs, mRNA expression for IFNG was screened for using three different mRNA concentrations. IFNG had a dose-dependent relationship with RNA concentration. Thus, we moved forward to investigate IFNG as a potential early marker for antigen specific activation in CD4+ T cells.
To increase the sensitivity of the assay, the incubation time was increased from 3 hours to an overnight. The additional incubation time increased IFNG expression by 7 fold, indicating improved activation of CD4 T cells. The RT-qPCR study was repeated using IL-2 and IFNG, resulting in a 20-fold and 50-fold increase in IFNG and IL-2 mRNA expression, respectively.

Conclusions:
RT-qPCR proves to be a sensitive and informative method for detecting antigen-specific T cell populations. The technique allows for rapid assessment of cytokine expression profiles, serving as indicators of T cell activation and function. The ability to detect these responses quickly and at various time points provides valuable insights into the kinetics of T cell activation. The observed donor variability underscores the importance of personalized approaches in immunotherapy, as individual patients may respond differently to the same antigen.

Discussion:
This RT-qPCR-based approach offers several unique advantages for evaluating T cell responses in the context of cancer immunotherapy. First, its sensitivity allows for the detection of CD4 responsiveness to specific antigens at an early stage of activation, a crucial capability when dealing with neo-antigens. Second, measuring multiple cytokines simultaneously provides a comprehensive view of the T cell response. Third, the rapid turnaround time of RT-qPCR makes it suitable for clinical applications where quick decision-making is essential. These unique advantages position RT-qPCR as a powerful tool in the field of cancer immunotherapy.
The technique could be valuable in screening and selecting potential neo-antigens for ACT therapies and monitoring T cell responses during treatment. The observed time-dependent increase in cytokine expression suggests that optimizing incubation periods may be critical for capturing the full extent of T cell responses. Furthermore, the differential expression patterns of IFNG and IL2 highlight the importance of assessing multiple cytokines at various timepoints to understand T cell functionality.

Acknowledgements (Optional):