(J-391) Investigate Short-Term Mechanosensing Response in T Cell Subsets

Engineered T cells have emerged as a potent tool to treat cancers and autoimmune diseases. Notably, chimeric antigen receptor (CAR) T cell therapy demonstrates remarkable success in treating hematological cancers. However, patients with long-term cancer such as chronic lymphocytic leukemia (CLL) often exhibit T cell exhaustion, leading to difficulties in cell expansion¹. The resulting low numbers of T cells reduce the efficacy of the therapy. 

T cell expansion relies on T cell receptor activation and a co-stimulatory signal, often achieved ex vivo using activating antibodies to CD3 and CD28 attached to a supporting material. Studies suggest that reducing the stiffness of these materials, such as replacing the conventional plastic supports with soft polydimethylsiloxane (PDMS), improves T cell expansion. However, variability in overall T cell mechanosensing among different donors complicates expansion. We hypothesize that the composition of T cell subsets plays a crucial role in the overall mechanosensing performance, considering demographic factors such as sex and age have been known to influence T cell subsets distribution^4,5. Our aim is to identify mechanosensitive subtypes and explore interactions between these populations through assays of short-term spreading, an early marker for T cell activation⁶. We hope to establish a system that tunes external mechanical environment to support T cell expansion and induces differentiation into preferred subsets, ultimately leading to personalized treatment to revolutionize immunotherapy.

T cell isolation: T cells were isolated from Leukapheresis packs from New York Blood Center and cultured in complete media consist of RPMI 1640 supplemented with FBS, HEPES, L-Glutamine, penicillin-streptomycin, and β-mercaptoethanol. PDMS fabrication and coating: PDMS with Young’s Modulus of 50kPa and 1MPa was fabricated using Sylgard 184 at a base-to-crosslinker ratio of 1:50 and 1:25, curing at 65˚C overnight. Cured PDMS was first coated with 1µg/mL biotinylated goat anti-mouse IgG at room temperature (RT) for 2 hours, blocked with 5% BSA at RT for 2 hours, and coated with 10µg/mL anti-CD3 (clone: OKT3) and 40µg/mL anti-CD28 (clone: 9.3) at 4˚C overnight. T cell spreading assay: T cells were seeded on glass supported antibodies coated PDMS, allowed to spread at 37˚C for 40 minutes. Cells were fixed and permeabilized using True-Nuclear Transcription Factor Buffer Set. Alexa Fluor (AF) 488 phalloidin, PerCP anti-human CD4, AF 647 anti-human CD45RA, and PE anti-human CCR7 were added in perm buffer to stain the cells. After staining, cells were washed by PBS and imaged under epi-fluorescence microscope. Cell morphologies were evaluated by ImageJ semi-automated segmentation. Flow cytometry: Purity of isolated T cells was verified by Live/Dead (L/D) Fixable Violet and FITC anti-human CD3 Antibody via FACSCanto I. T cells were also stained with L/D FITC, PerCP anti-human CD4, AF 647 anti-human CD45RA, and PE anti-human CCR7 and assessed via Cytek Aurora to collect baseline composition of T cell subsets. Statistics: Statistical significance was determined using two-way ANOVA with multiple comparisons.

Results and Discussion: T cells from a donor that has been previously verified for significant mechanosensing response was used for the study (Fig 1D). Cell subsets spreading on PDMS substrates were identified via surface markers staining (Fig 1A, 1C). Initially, T cells were characterized into CD4+ or CD8+ subsets, with CD4+ T cells demonstrating a more pronounced increase in spreading area when seeded on softer substrates, indicating a higher mechanosensing response in CD4+ T cells (Figure 1E). Within the CD4+ T cell population, effector memory T cells (T_EM) exhibited the highest increase in spreading area, while central memory T cells (T_CM) showed minimal response (Figure 1F). Among the CD8+ T cell subsets, no significant different in mechanosensing has been detected. However, CD8+ effector T cells (T_E) and T_EM had a greater average spreading area than CD8+ naïve T cells (T_N) and T_CM (Figure 1G). Such difference in spreading area among subsets may be attributed to the presence of distinct receptors on cell surface, leading to varied levels of mechanotransduction that regulate cellular cytoskeleton contraction. 

Conclusion: Preliminary data shows that different T cell subsets display different level of mechanosensing response. For example, CD4+ T_EM cells have strong mechanosensing response comparing with other subsets. We will also conduct repetitive experiments on this donor and expand to a broader range of donors with diverse demographics to gather more data and obtain valuable insight into mechanosensing within distinct T cell subsets. Ultimately, we aim to utilize the mechanosensing properties in subsets to develop a platform that induces T cell differentiation into preferred subsets and optimize ex vivo T cell expansion to enhance T cell-based immunotherapy.

[1] Riches, J. C., et. al. (2013) “T cells from CLL patients exhibit features of T-cell exhaustion but retain capacity for cytokine production,” Blood. 121(9): 1612–1621.
[2] Kim, S.T. et. al. (2009) “The αβ T Cell Receptor Is an Anisotropic Mechanosensor,” J Biol Chem. 284(45):31028-37.
[3] O’Connor RS, et al. (2012) “Substrate rigidity regulates human T cell activation and proliferation,” J Immunol. 189(3):1330–1339.
[4] Abdullah, A., et. al. (2012) “Gender effect on in vitro lymphocyte subset levels of healthy individuals,” Cell. Immunol. 272: 214-219.
[5] Klein, S. and Flanagan, K. (2016) “Sex differences in immune responses,” Nat. Rev. Immunol. 16: 626–638.
[6] Wahl, A., et. al. (2019) “Biphasic mechanosensitivity of T cell receptor-mediated spreading of lymphocytes.” PNAS, 116 (13) 5908-5913.