Fig. 1: Limited cross-reactivity of the low-affinity A4Y peptide.
From: Regulatory T cells define affinity thresholds for CD8+ T cell tumor infiltration
A A cartoon illustrating antigen presentation of the low-affinity A4Y glycoprotein peptide (KAVANFATM) and p33 (KAVYNFATM). In A4Y variant, tyrosine (Y) is substituted by alanine (A) at position 4. B 1% agarose gel electrophoresis image, with Lane 1, DNA ladder; Lane 2, Qβ(ssRNA); Lane 3, Qβ(ϕ) empty; Lane 4, Qβ(CpGs). ODN 1668, TLR9 was used in all experiments. The top part of the gel is stained with SYBR Safe, while the bottom part is stained with Coomassie Blue. C SDS-PAGE stained with Coomassie Blue, with Lane 1, protein marker; Lane 2, Qβ(CpGs) at 14 kDa; and Lane 3, Qβ(CpGs) derivatized with a dibenzocyclooctyne bifunctional cross-linker (DBCO), Lanes 4 and 5 show coupling to p33 or A4Y peptides respectively. Red box indicates Qβ(CpGs) monomer before and after derivatization with DBCO and coupling to peptides. Black arrows indicate the coupled peptides to a Qβ monomer. D, E Percentage of A4Y+ and p33+ cells within CD8+ T cells in the spleen, measured 8 days post-vaccination with Qβ control, Qβ-A4Y or Qβ-p33. F, G Representative flow cytometry plots of (D, E). H Percentage of cross-reactive specific T cells. I, J Number of IFN-γ producing A4Y+ and p33+ cells within CD8+ T cells in the spleen per 2 × 105 splenocytes, measured 8 days post-vaccination with Qβ, Qβ-A4Y or Qβ-p33. K Representative flow cytometry plots of (I). L Experimental setup for the assessment of killing in vivo. M, N Percentage of CFSE labeled splenocytes, pulsed with A4Y or p33 peptide in mice vaccinated with Qβ, Qβ-A4Y, or Qβ-p33. O, P Percentage of in vivo lytic activity. Please note that negative lytic activity normally occurs in this type of analysis. Q Histograms showing the count of CFSE-pulsed splenocytes. Statistical analysis with Student’s t-test or one-way ANOVA. The sample size for each group was n = 3.
