Age and Sex as Biological Variables in Ex Vivo CD3/CD28-Mediated T Cell Activation

Introduction: : T cells are a crucial part of adaptive immunity against cancer. Many current T cell therapies are based on adoptive cell transfer in which ex vivo activation and expansion of T cells are necessary to generate an immune response strong enough to combat cancer. This process involves harvesting T cells from the patient, often treating or genetically modifying them, expanding the population, and reinfusing them into the patient. One example of this is ex vivo treatment of T cells with fluid shear stress to enhance their activation [1]. The expansion of cells is typically done using artificial antigen presenting cells such as Dynabeads that are coated with anti-CD3 and -CD28 antibodies to trigger T cell activation and proliferation through T cell receptor signaling. However, this is an expensive and time-consuming process where variability of T cell response across patients represents unpredictability [2]. Specifically, the degree of proliferation and differentiation into different effector subtypes can vary from one patient to another. To better predict the success of these therapies, we have investigated trends across age or sex in how T cells respond to activation using CD3/CD28 stimulation.

Materials and

Methods: : Buffy coat from healthy male and female donors between 17 to 69 years old was acquired. Peripheral blood mononuclear cells (PBMCs) were isolated via density separation and frozen. Once read for analysis, PBMCs were thawed and T cells were isolated by magnetic separation using negative selection. To assess proliferation, cells were stained with CellTrace™ CFSE Cell Proliferation Kit and incubated with anti-CD3/CD28 coated Dynabeads for 4 days. CSFE fluorescence was then measured using flow cytometry to track the parent and daughter populations. T cell differentiation was also determined by quantification of IL2 and IFN-γ. Donor T cells were incubated with Dynabeads for 24 hours. GolgiPlug protein transport inhibitor was then added to culture for 4 hours after which cytokine buildup in the cells was quantified using flow cytometry to determine cell signaling and effector function. Additionally, viability was measured simultaneously to proliferation and differentiation studies.

Results, Conclusions, and Discussions:: Ex vivo activation of human donor T cells resulted in a variable response among all donors. There was a large spread in the percentage of CD4+ T cells that produced IL2 and IFN-γ. However, when these populations were compared by age or sex there was no significant difference in the emergence of these cytokine producing cells. Male samples showed more variability compared to female and donors younger than 41 years old had more variability compared to those older than 57 years. Cell viability was not affected significantly in any of the groups. These results suggest that discernable trends by age or sex in ex vivo T cell activation may arise with the analysis of a larger sample size. It also indicates that there could be other demographic factors to consider when assessing a patient’s response to this treatment.

T cell expansion and cytokine production are important indicators for T cell response and functions that demonstrate their potential to successfully combat cancer. A greater population of IL2 and IFN-γ producing cells demonstrate stronger signaling for T cell expansion and recruitment of cytotoxic immune cells. Considering the variable response in these metrics to ex vivo T cell therapies, future studies should be done to further assess the role of age and sex as well as other possible predictors for the success of ex vivo T cell therapies.

Acknowledgements and/or References (Optional):: [1] J. M. Hope et al., “Fluid shear stress enhances T cell activation through Piezo1,” BMC Biol, vol. 20, no. 1, Dec. 2022. [2] Chun Jimmie Ye et al. Intersection of population variation and autoimmunity genetics in human T cell activation.Science345,1254665(2014).