APC Application Note

Opportunities

Enhanced MHC-I presentation: Mechanoporation of antigens directly into the cytosol can increase the efficiency of MHC-I presentation, even in professional APCs, by as much as 1,000-fold. Presented MHC-I peptides are derived from cytosolic proteins and peptides, so by delivering antigens directly into the cytosol, mechanoporation shuttles antigens more efficiently into the MHC-I pathway. While some professional APCs are capable of a process termed cross-presentation, by which a fraction of antigens escape endosomes to enter the cytosol, the efficiency of this process pales in comparison to antigens mechanoporated directly into the cytosol.
MHC-I presentation by non-classical APCs: By delivering directly into the cytosol, mechanoporation enables MHC-I presentation of delivered peptides across a wide array of cell types, obviating the need for laborious generation of professional APCs from rarer cell types.
Increased sensitivity to low concentrations of antigen: Given the efficiency of directing antigens into the MHC-I pathway, lower concentrations of antigen are required to detect peptide-MHC-I loading.
Potential for antigen screening in lysates: Given the increased sensitivity and ability to deliver proteins/peptides from lysates, it may be possible to screen lysates for presented peptides or reactive T cells.

Results Obtained

Delivery of model antigens and assessment of MHC-I presentation in mouse and human systems
Limits of delivery parameters actively being investigated as part of Portal’s work with DARPA

Mechanoporation enables 1,000-fold increase in efficiency

We demonstrated that mechanoporation enables a 1,000-fold increase in efficiency of MHC-I presentation by professional antigen presenting cells. Mouse bone marrow-derived dendritic cells (BMDCs) were incubated or mechanoporated with various concentrations of the model antigen, ovalbumin (OVA). These BMDCs were then co-cultured with OVA-specific CD8+ T cells (OT-Is) overnight and MHC-I presentation of OVA peptides was monitored by CD69 upregulation on OT-I T cells, a measure of T cell activation.

Fig 1: Mouse bone marrow-derived dendritic cells (BMDCs) were mechanoporated or incubated with the model antigen ovalbumin (OVA). They were then co-cultured with OVA-specific CD8+ T cells (OT-Is) overnight and MHC-I presentation of OVA peptides was monitored by CD69 upregulation on OT-I T cells, a measure of T cell activation.

Enables MHC-I presentation by a broad array of cell types

We ran experiments to show that mechanoporation enables MHC-I presentation by a broad array of cell types, including non-classical APCs. We mechanoporated mouse splenocytes with or without OVA. At the indicated time points, cells were stained with an antibody (25-D1.16) recognizing H-2Kb presented OVA epitope, SIINFEKL. Then cell types were distinguished by phenotypic markers and flow cytometry. Click through to see data for all the cell types!

Fig 3: Mouse T cells mechanoporated with ovalbumin and stained with an antibody (25-D1.16) recognizing H-2Kb presented OVA epitope, SIINFEKL

Fig 4: Mouse B cells mechanoporated with ovalbumin and stained with an antibody (25-D1.16) recognizing H-2Kb presented OVA epitope, SIINFEKL

Fig 5: Mouse NK cells mechanoporated with ovalbumin and stained with an antibody (25-D1.16) recognizing H-2Kb presented OVA epitope, SIINFEK

Fig 6: Mouse monocytes cells mechanoporated with ovalbumin and stained with an antibody (25-D1.16) recognizing H-2Kb presented OVA epitope, SIINFEKL

Mechanoporation facilitates efficient MHC-I presentation of a variety of antigens

We used Mechanoporation to facilitate efficient MHC-I presentation of a variety of antigens by human PBMCs. Human peripheral blood mononuclear cells (PBMCs) were mechanoporated with the indicated antigens in mRNA form. PBMCs were then co-cultured with human antigen-specific T cells. For CMV p65 and Influenza M1, primary CD8+ T cells from HLA-A*02 donors were expanded against CMV pp65(495-503) or Influenza M1(58-66) epitopes and used as responders. IFNg production after overnight culture was measured in the supernatant by ELISA. For HPV16 E6, HPV16 E7, and KRAS G12V reactivity, Jurkat NFAT reporter cells were transduced with TCRs specific for E6(29-38) and E7(11-19), or KRAS G12V(8-16) and used as responders. Luciferase production in the supernatant was measured by spectrophotometer.

Fig 7: PBMCs mechanoporated with CMV pp65 mRNA were co-cultured overnight with HLA-A*02 CMV pp65(495–503)–specific CD8⁺ T cells. IFNγ in supernatants (y-axis, pg/mL) was measured by ELISA: mechanoporated PBMCs produced ~9×10³ pg/mL IFNγ, while untreated PBMCs were near background. This figure shows efficient MHC-I presentation of pp65 after mechanoporation.

Fig 8: PBMCs mechanoporated with Influenza M1 mRNA (“M1 mRNA Squeeze”) were co-cultured overnight with with human antigen-specific T cells. IFNγ in supernatants (y-axis, pg/mL) measured by ELISA shows robust production from mechanoporated PBMCs (~3×10³ pg/mL) versus near-background from untreated cells. This indicates efficient MHC-I presentation of M1 after mechanoporation.

Fig 9: PBMCs mechanoporated with E6 mRNA were co-cultured overnight with Jurkat NFAT reporter cells expressing an E6(29–38)–specific TCR. Supernatant luminescence (RLU) showed strong activation from mechanoporated PBMCs (~1.2×10⁴ RLU) versus low signal from untreated PBMCs. This demonstrates efficient MHC-I presentation of E6 following mechanoporation.

Fig 10: PBMCs mechanoporated with E7 mRNA were co-cultured overnight with Jurkat NFAT reporter cells expressing an E7(11–19)–specific TCR. Supernatant luminescence (RLU) shows robust activation from mechanoporated PBMCs (~3×10⁴ RLU) versus near-background from untreated cells. This indicates efficient MHC-I presentation of HPV16 E7 after mechanoporation.

Fig 11: PBMCs mechanoporated with G12V mRNA were co-cultured overnight with Jurkat NFAT reporter cells bearing a KRAS G12V(8–16)–specific TCR. Supernatant luminescence (RLU) shows strong activation from mechanoporated PBMCs (~3×10⁴ RLU) versus low background from untreated cells. This demonstrates efficient MHC-I presentation of KRAS G12V after mechanoporation.

Original Sources:

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