Fig 2: NK cell analysis of blood samples taken 36 h post AOaV-1 administration to mice previously i.m. administered AAV-3TSR (1 × 1011 vg), Fc3TSR (1 × 1011 vg) or AAV-Bevacizumab (1 × 1010 vg). Non-terminal retro-orbital blood samples were obtained 36 h post i.v. administration of 1 × 108 PFU AOaV-1. Red blood cells were lysed, and the remaining cells were stained and analyzed via flow cytometry. (A) Percentage of CD45+ cells that are NK cells. Percentage of NK cells expressing (B) the early activation marker CD69, (C) the immune checkpoint protein programmed cell death protein 1 (PD-1), (D) the immune checkpoint protein programmed death ligand 1 (PD-L1), (E) the cytotoxic activating receptor NKG2D, (F) the natural cytotoxicity receptor NKp46, (G) the NK functional marker CD107a, (H) the serine protease Granzyme (B,I) IFN-?. AOaV-1, avian orthoavulavirus-1; AAV, Adeno-associated virus; PBS, phosphate-buffered saline mock control group; 3TSR, thrombospondin-1 type I repeats; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant. Error bars denote standard deviation.

Fig 3: Robust Immunization Overcomes Peripheral Tolerance of Dysfunctional High-Affinity Self-Specific CD8 T Cells(A) IFN-? and TNF-a production by OTI from small intestine intraepithelial lymphocyte (IEL) compartment after ex vivo peptide stimulation. Left (tolerant): 60 days after adoptive transfer in iFABP-Ova hosts. Right (effector): day 7 after VSV-Ova in C57BL/6 hosts. Plots are gated on OTI.(B) iFABP-Ova mice were (+VSV-Ova) or were not (naive) infected with VSV-Ova 72 days after transfer of OTI. IELs were analyzed 359 days after OTI transfer. Plots are gated on i.v. negative CD8+ T cells. Numbers represent the frequency in gate.(C) OTI in PBL after day 0 of 1° (VSV-Ova) infection in B6 and iFABP-Ova mice. IFABP-Ova mice were infected 60 days after OTI transfer. B6 mice were infected 2 days after OTI transfer. The arrows indicate immunization (1° VSV-Ova; 2° LM-Ova; 3° VV-Ova). Solid black, B6; dashed gray, iFABP-Ova. Numbers, fold expansion of OTI at day 7 after 1° and day 7 after 3°, relative to day 0.(D) Number of OTI without infection (naive) and at days 30–40 post-3° in the IEL, spleen (SPL), and mLN. Filled, B6; open, iFABP-Ova; square, unimmunized; circle, after 3°. Numbers on graph, fold expansion from naive to 3°. Naive B6 IELs not included, as naive T cells are not found in NLTs.(E) IFN-? and TNF-a production by splenic OTI 7 days after 3° in B6 and iFABP-Ova after ex vivo peptide stimulation. Plots are gated on OTI.(F) Weight of B6 and iFABP-Ova mice after 3°. Black, B6; red, iFABP-Ova.(G) Geometric mean fluorescence intensity (GMFI) of Kb-SIINFEKL tetramer staining of OTI in PBLs of iFABP-Ova mice at indicated time points, normalized to wild type.(H) Histograms of Kb-SIINFEKL tetramer staining of the OTI enumerated in (G). Black, B6 OTI. Red, iFABP-Ova OTI.(I) Correlation of GMFI of Kb-SIINFEKL tetramer staining and GMFI PD-1 on OTI from iFABP-Ova mice day 6 after 3° in PBL. R2 value = 0.04275.Representative of at least 2 experiments totaling 3–10 animals per group. *p = 0.037, **p = 0.007, ***p = 0.0002. Error bars indicate means ± SEMs.See also Figures S1 and S2.

Fig 4: Sequential Immunization Drives Avidity Maturation of Functional, Endogenous Self-Specific CD8 T CellsB6 or iFABP-Ova mice were subjected to Ova immunization.(A) PD-1 and granzyme B expression by Kb-SIINFEKL-specific splenocytes 35 days post-3°. Histograms are gated on Kb-SIINFEKL-specific CD8 T cells from iFABP-Ova (dashed) and B6 (solid) or CD44lo CD8 T cells (gray filled).(B) GMFIs of indicated MHC I tetramer staining in splenocytes of iFABP-Ova (white) or B6 (black) mice at least 30 days after 3° VV-Ova.(C) Splenocyte IFN-? and TNF-a production with ex vivo peptide stimulation =30 days after 1°, 2°, or 3°. Plots are gated on CD8 T cells.(D) In vivo killing of SIINFEKL-pulsed target cells 30 days post-1° or 3°. Black, B6; white, iFABP-Ova.(E) Enumeration of splenocytes binding Kb-SIINFEKL tetramer or producing IFN-? =30 days after 1°, 2°, or 3°. Black, tetramer+; white, IFN-?. The dotted line represents CD8 T cells readily detectable in B6 at 1° with Kb-SIINFEKL tetramers.(F) Kb-SIINFEKL-specific CD8 T cell responses detected either with MHC I tetramer staining (Tet+) or cytokine assay (IFN-?+) in iFABP-Ova mice after 2° as a function of Kb-SIINFEKL tetramer-binding cells in B6 mice after 1°.(G) Percentage of maximum IFN-? produced by splenic CD8 T cells = 30 days after 2° or 3°. GMFI of IFN-? production at 2° or 3° normalized to maximum GMFI of IFN-? at matching time point. Black, B6; white, iFABP-Ova.Representative of at least 2 experiments totaling 4–12 animals per group. *p < 0.037, **p = 0.009, ***p = 0.0006. Error bars indicate means ± SEMs, except in (B) where it is SDs.See also Figure S3.