(M1330-05-25) Internalization and Trafficking Pathway of T-Cell Receptor Mimic Antibodies (TCRm) RL6A in Human Brain Endothelial Cells at the Blood Brain Barrier (BBB)

Purpose: T-cell receptor mimic antibodies (TCRm), a novel class of antibodies, recognize specific peptides in the context of MHC (HLA in humans) class I molecules with higher specificity and binding affinity than T-cell receptor (TCR). HLA class-I are natural surface biomarkers that reflect each cell’s distinct intracellular proteome for extracellular immune recognition. Therefore, unique expression patterns of peptide/MHC complexes may allow to specifically target different cell types using TCRm as immunotherapeutic agents. RL6A is a TCRm specific to the endogenous peptide/MHC complex, YLLPAIVHI/HLA-A2 (YLL-A2). This peptide is derived from human P68 RNA helicase. The YLL-A2 complex is extensively expressed by human breast cancers and other tumors, but it may also be a target on vascular endothelial cells. Our lab previously showed binding and internalization of RL6A by primary human brain-derived endothelial cells and by the human brain endothelial cell line hCMEC/D3 [1]. Here we studied internalization of RL6A in induced Pluripotent Stem Cells (iPSC) derived human brain microvascular endothelial cells (hBMEC) compared to hCMEC/D3 cells. Also, we performed trafficking study of RL6A in hCMEC/D3 cells.

Methods: We analyzed the amount of surface-bound TCRm antibodies on the human brain endothelial cells using flow cytometry. hCMEC/D3 cells and hBMEC were incubated with RL6A or isotype control antibodies for 30 min at 4°C, followed by incubation at 37°C to initiate the internalization. We studied RL6A binding and internalization with or without prior stimulation of the cells by interferon-gamma (IFN‐γ). Confocal Z-stack images were acquired on a Leica Stellaris 8 FALCON STED system to characterize the trafficking pathway of fluorescent dye-labelled RL6A in hCMEC/D3 cells. We analyzed RL6A colocalization with the endosomal markers ARF6, Rab5, and EEA1. AIVIA 3D software was used to obtain Pearson’s correlation coefficient as a measure of colocalization between RL6A and these markers. Further, 3-D Stimulated Emission Depletion (STED) images were subject to object-based colocalization analysis.

Results: hCMEC/D3 cells internalized RL6A in a biphasic pattern, with rapid internalization at early time points ( >30% internalized within 10min) followed by a slower phase. The signals for surface-bound RL6A were significantly reduced after 120min endocytosis initiation by iPSC-derived hBMEC. Furthermore, the mean fluorescence intensity (MFI) for surface-bound RL6A increased by around two-fold for the interferon-gamma (IFN‐γ) treated cells in both hCMEC/D3 and hBMEC cells. RL6A and the small GTPase ARF6 colocalized highest at 30 min and 120 min. Rab5 showed the highest Pearson’s value at 30 min. Object-based colocalization analysis indicated that EEA1 is involved in the trafficking of RL6A at all time points with a peak at 10 min. Large EEA1 vesicles showed a higher degree of colocalization compared to small vesicles at all time points.

Conclusion: RL6A binding and internalization showed the similar pattern in both hCMEC/D3 and hBMEC. TCRm RL6A trafficking is consistent with an ARF6 associated pathway of clathrin-independent trafficking that has been described for MHC class I in other cell types [2-4]. Internalization occurs mostly within 30. IFN‐γ treatment upregulated the expression level of the YLL-A2 complex and increased the RL6A binding and internalization, although it did not change the dynamics of internalization. Increased RL6A colocalization with Rab5 at 30 min and 60 min suggests endosome fusion happening in this time frame. High colocalization of RL6A with ARF6 at 120min might be the result of ARF6 involvement in the recycling pathway. The object-based analysis allowed better quantitative colocalization analysis.

References: 1. Bhattacharya, R., et al., A novel vascular targeting strategy for brain-derived endothelial cells using a TCR mimic antibody. J Cell Physiol, 2010. 225(3): p. 664-72.
2. Donaldson, J.G., D.L. Johnson, and D. Dutta, Rab and Arf G proteins in endosomal trafficking and cell surface homeostasis. Small GTPases, 2016. 7(4): p. 247-251.
3. Mayor, S., R.G. Parton, and J.G. Donaldson, Clathrin-independent pathways of endocytosis. Cold Spring Harb Perspect Biol, 2014. 6(6).
4. Porat-Shliom, N., R. Weigert, and J.G. Donaldson, Endosomes derived from clathrin-independent endocytosis serve as precursors for endothelial lumen formation. PLoS One, 2013. 8(11): p. e81987.


[Figure 1] MFI of cell surface-bound RL6A with and without 24 hr IFN‐γ treatment using hCMEC/D3 cells. A) Surface-bound antibodies after the binding step of 30 min at 4°C. IFN‐γ significantly increased the amount of RL6A binding on the cell surfaces. Isotype IgG2a was used for control. There was a significant increase in RL6A binding compared to isotype control. B) Comparison of surface-bound antibodies after the binding step of 30 min at 4°C and after the internalization step of 120 min at 37°C. For both IFN‐γ treated and untreated groups, there was a significant reduction of surface-bound RL6A after 120min of the internalization step. Isotype control didn’t significantly change between 0 min and 120 min, while BB7.2 showed a significant decrease.


[Figure 2] Pearson’s correlation coefficient values between RL6A and endosomal markers (A) ARF6 and (B) RAB5. A) ARF6 and RL6A showed the highest colocalization rate at 30 min and 120 min. B) RAB5 colocalized with RL6A mostly at 30min and slowly decreased by time until 120 min.


[Figure 3] EEA1 and RL6A colocalization analysis by (A) Pearson’s correlation coefficient and (B) object-based colocalization. A) Pearson’s value didn’t significantly change over time except between 60 min and 120 min. B) Object-based analysis showed more dynamic changes in colocalization between EEA1 and RL6A. Highest colocalization rate was observed at 10min and decreased over time.