Fig 2: IL-2 supports T cell viability and sustained activation in co-culture media.A Schematic diagram illustrating the strategy of T cell isolation from human peripheral blood mononuclear cells (PBMCs) and polyclonal activation using anti-CD3/CD28-coupled dynabeads. Some parts of the diagram created with BioRender.com under a licensed academic agreement. Winner, B. (2024), and some created in Microsoft PowerPoint. B Validation of purity and activation of T cells prior to co-culture with organoids. Left: Successful T cell isolation was revealed by roughly 95% of CD3+ cells. Middle: Efficiency of ex vivo T cell activation determined by CD25 expression in non-activated and activated T cells after 48 hours (h) of incubation with CD3/CD28 beads. “Activated (A)”: T cells were activated for 48 h using CD3/CD28 dynabeads. “non-activated (nonA)”: T cells were incubated for 48 h without activation stimuli in the presence of IL-2. Right: Successful activation of T cells was confirmed by around 100% of activated (A) T cells expressing CD25 in contrast to non-Activated (nonA) T cells. C Flow cytometry-based T cell viability and activation assessment in different conditions. Contour and Dot plots of T cells cultured for 7 days with CD3/CD28 beads in hMO medium (hMOM) or Serum-Free Lymphocyte Medium (SFLM) with or without IL-2. D Quantification of T cell viability (live/dead staining) and activation (CD3+/CD25+ double positivity) demonstrates that in both media, IL-2 supports a more sustained activation and survival of T cells. *p < 0.05, ****p < 0.00005, two-way ANOVA.

Fig 3: T cell infiltration correlates with cell death and neuronal loss in hMO tissue.A Increased cell death in hMO co-culture with T cells (+T cells) in comparison to hMO cultured without T cells (no T cells). Left: Representative immunostaining of CD3 (green) and TUNEL (orange). Scale bar = 200 μm. Right: Quantitative comparison of cell death (% TUNEL+ cells) in hMO co-cultured with or without T cells. *p < 0.05, Student’s t test. B Correlation between T cell presence and cell death in hMO tissue. Scatter plot showing the relationship between T cell presence (% CD3+ cells) and cell death (% TUNEL+ cells) in hMO. A significant correlation was observed (r = 0.8667, **p = 0.0045, Spearman’s correlation, two-tailed). C Majority of TUNEL signal (examples indicated by arrows) localizes in MAP2+ neuronal somas. Representative immunostaining of TUNEL (yellow) and MAP2 (red). Scale bar = 20 μm. D Decreased MAP2 signal in hMO co-cultured with T cells in comparison to hMO cultured without T cells. Left: Representative immunostaining of CD3 (green) and MAP2 (red). Scale bar = 200 μm. Right: Quantitative comparison of MAP2+ neurons (rel. MAP2 signal) in hMO cultured with or without T cells. *p < 0.05, Student’s t test. E Correlation between T cell presence and MAP2+ neurons in hMO tissue. Scatter plot showing the relationship between T cell presence (% CD3+ cells) and rel. MAP2 signal in hMO. A significant negative correlation was observed (r = −0.8303, **p = 0.0047, Spearman’s correlation, two-tailed). F T cells localize in the vicinity of MAP2+ neurons (examples indicated by arrows). Representative immunostaining of CD3 (green) and MAP2 (red). Scale bar = 50 μm. Quantitative flow cytometry analysis of G CD4 and CD8 fractions, H cytolytic and degranulation markers CD107a and granzyme B (GZMB), and I pro-inflammatory (IFN‐γ, IL-2, IL-17, TNF-α) and anti-inflammatory (IL-10) cytokines expressed by infiltrating T cells.

Fig 4: Schematic diagram illustrating the strategy of development and usage of T cell-midbrain organoid co-culture system.As a first step, different co-culture conditions were tested. hMOM with IL-2 was shown to be optimal for viability of both, hMO and T cells. As a second step, T cell infiltration into hMO and T cell-neuron interactions were studied. T cells were shown to induce cell death and MAP2+ neuronal loss. As a third step, age- and region-specific susceptibility of brain organoids to T cell infiltration was revealed. Day 60 aged hMO were shown to be more vulnerable to T cell infiltration and T cell-driven neurotoxicity. Thus, the 3D co-culture model developed in this study provides a versatile platform for investigating immune-neural interactions in PD: by incorporating PD-specific T cells and brain organoids derived from PD patients’ stem cells, it enables the examination of T cell infiltration into hMO, spatial colocalization and cell-type specificity of T cell interactions within organoid tissue, T cell-driven effects, adhesion mechanisms facilitating T cell infiltration, and functional characterization of T cells and their subsets infiltrating the organoids. Figure is created with BioRender.com under a licensed academic agreement. Winner, B. (2024) https://BioRender.com/v54o777.

Fig 5: Assessment of the hMO viability in different conditions.A Schematic diagram illustrating the overall strategy of testing the effect of 4 different conditions on hMO. Immunocytochemistry was used for evaluation of cell death and neuronal survival by the TUNEL assay and MAP2 staining, respectively. Some parts of the diagram created with BioRender.com under a licensed academic agreement. Winner, B. (2024), and some created in Microsoft PowerPoint. B Quantification of cell death (% TUNEL+ cells) within organoid tissue. Increased TUNEL signal was detected in Serum-Free Lymphocyte Medium (SFLM) without IL-2, suggesting that SFLM itself causes cell death in hMO. C Comparison of hMO viability after culturing them for 7 days in four different conditions. Up: Representative cryosections of hMO stained for MAP2 (purple), TUNEL (yellow) and DAPI (blue). Scale bar = 500 μm. Bottom: A zoom-in view. Yellow scale bar = 100 μm.