Figure 2. CD44lo and CD44hi CD4+ T-cells binding immunodominant, subdominant, and allogeneic pMHC increase with time.
Representative plots of CD4+ T-cells bound to (A and B) E641:I-Ab, (C and D) OVA:I-Ab, and (E and F) MCC:I-Ek tetramers in adult (top) and old (bottom) mice. Absolute number of CD4+ CD44lo (left Y-axis) and CD4+ CD44hi (right Y-axis) T-cells bound to (G) E641:I-Ab, (H) OVA:I-Ab, or (I) MCC:I-Ek tetramers only enumerated after dump tetramer analysis (‘Materials and methods’), or those binding (J) E641:I-Ab + OVA:I-Ab, (K) OVA:I-Ab + MCC:I-Ek, or (L) E64:I-Ab + MCC:I-Ek tetramers in combination enumerated after both dump and two-color tetramer analysis (‘Materials and methods’). Bars indicate median (*p < 0.05, **p < 0.005, ***p < 0.0001, ns = non-significant; Mann–Whitney). Fold change (Δ) in means between adult and old is shown. Results are from three experiments with 4 mice/group.
Figure 2—figure supplement 1. Tetramer validation on T-cell hybridomas.
Dot plots showing E641:I-Ab (left panel), OVA: I-Ab (middle panel), and MCC:I-Ek (right panel) tetramer bound to (A) parental 58α−β− TCR-negative hybridomas or those transduced to express the (B) OT-II TCR specific for OVA: I-Ab, or the (C) 5c.c7 or (D) 2B4 TCRs specific for MCC:I-Ek. Each tetramer was made with streptavidin-PE and streptavidin-APC for two-color analysis as labeled on axis.
Figure 2—figure supplement 2. Tetramer validation for in vivo primed CD4+ T-cells.
C57BL/6 mice were immunized with 50 µg of the indicated peptide in 50 µl CFA on each side of the base of the tail or left unprimed. Spleen and lymph nodes cells were harvested on day 7-post immunization and incubated with E641:I-Ab, OVA:I-Ab, and MCC:I-Ek tetramers. No tetramer enrichment was performed. Contour plots show tetramer vs CD44 for (A–C) unprimed (D–F) E641+CFA immunized or (G–I) OVA+CFA immunized mice. Numbers indicate percent of CD44hi tetramer+ CD4+ T-cells. Results represent a single experiment with one mouse per condition. (J) Amino acid sequences of peptides contained in I-Ab and I-Ek tetramers used in this study. The bold sequences indicate the core nonamer peptide epitopes as predicted by Zhu et al. (2003); Birnbaum et al. (2012); Nelson et al. (2015). OVA 326–338 is from chicken ovalbumin, Env 641–655 is from West Nile Virus, and MCC 88–103 is from moth cytochrome c. The putative TCR contact residues are highlighted in red.
Figure 2—figure supplement 3. WNV-specific CD4+ T-cells increase over the lifespan.
Representative gating strategy for CD4+ tetramer+ T-cells via (A) forward and side scatter, (B) CD3+ CD8− T-cells, (C) CD3+ CD4+ T-cells, and (D) E641:I-Ab+ CD4+ T-cells in the tetramer enrichment bound fraction from adult and old mice. (E) Absolute number of E641:I-Ab+ CD4+ T-cells derived from pooled spleen and lymph node cells from unprimed adult or old mice after E641:I-Ab tetramer enrichment using anti-FP magnetic beads. Horizontal bar indicates median (*p < 0.05; Mann–Whitney). Results are shown from one of two similar experiments (4 mice/group).
Figure 2—figure supplement 4. E641 is immunodominant to OVA.
C57BL/6 mouse was co-immunized with 25 µg E641 + 25 µg OVA + 50 µl CFA on each side of the base of the tail. On day 7 post immunization, pooled spleen and lymph node cells were harvested and stained with E641:I-Ab and OVA:I-Ab tetramers. No tetramer enrichment was performed. (A) Contour plot showing E641:I-Ab vs OVA:I-Ab tetramer+ CD4+ T-cells. (B) CD44 vs E641:I-Ab tetramer+ CD4+ T-cells. (C) CD44 vs OVA:I-Ab tetramer+ CD4+ T-cells. Number indicates percent CD44hi tetramer+ CD4+ T-cells of total CD4+ T-cells. Results are from a single experiment.
Figure 2—figure supplement 5. Gating scheme for identification of tetramer+ cells.
Representative gating scheme for identifying tetramer+ cells for Figures 2, 4. (A) Forward vs side scatter. (B) CD3+ CD8− T-cells. (C) CD4+ T-cells. After anti-His magnetic enrichment, the tetramer+ gate was set on the unbound fraction to identify (D) E641:I-Ab+, (E) OVA:I-Ab+, and (F) MCC:I-Ek+ CD4+ T-cells. (G) Additional gating on E641:I-Ab+ CD4+ T-cells in the bound fraction to further identify (H–J) tetramer single+, double+, and triple+ CD4+ T-cells (E = E641:I-Ab+, O = OVA:I-Ab+, and M = MCC:I-Ek+).
Figure 2—figure supplement 6. Poly-specific cells form a very small fraction of a particular total tetramer+ population.
Percent tetramer single+, double+ and triple+ CD4+ T-cells of (A) Total E641:I-Ab+, (B) Total OVA:I-Ab+, and (C) Total MCC:I-Ek+ CD4+ T-cells in adult (left) and old (right) mice. Horizontal bar indicates median. Data are from three experiments with 4 mice/group.
Figure 2—figure supplement 7. Two-color analysis of E641+OVA polyspecific cells ± dump tetramer exclusion.
CD4+ T-cells from pooled spleen and lymph nodes from unprimed adult and old mice after tetramer enrichment using anti-His magnetic beads. Shown are representative plots of the CD4+ T-cells binding E641:I-Ab + OVA:I-Ab tetramers pre (left) and post (right) gating out the dump, MCC:I-Ek, tetramer+ cells in (A) adult and (B) old mice. Absolute number of CD44lo (left Y-axis) and CD44hi (right Y-axis) CD4+ T-cells binding E641:I-Ab + OVA:I-Ab tetramers (C) pre and (D) post gating out the dump MCC:I-Ek tetramer+ cells. Horizontal bar indicates median (**p < 0.005, ***p < 0.0001, ns = non-significant; Mann–Whitney). Δ: Fold change in average numbers from adult to old mice. Data are from three experiments with 4 mice/group.
Figure 2—figure supplement 8. Two-color analysis of OVA+MCC polyspecific cells ± dump tetramer exclusion.
CD4+ T-cells from pooled spleen and lymph node from unprimed adult and old mice after tetramer enrichment using anti-His magnetic beads. Shown are representative plots of the CD4+ T-cells binding OVA:I-Ab + MCC:I-Ek tetramers pre (left) and post (right) gating out the dump, E641:I-Ab, tetramer+ cells in (A) adult and (B) old mice. Absolute number of CD44lo (left Y-axis) and CD44hi (right Y-axis) CD4+ T-cells binding OVA:I-Ab + MCC:I-Ek tetramers (C) pre and (D) post gating out the dump, E641:I-Ab, tetramer+ cells. Horizontal bar indicates median (**p < 0.005, ***p < 0.0001, ns = non-significant; Mann–Whitney). Δ: Fold change in average numbers from adult to old mice. Data are from three experiments with 4 mice/group.
Figure 2—figure supplement 9. Two-color analysis of E641+MCC polyspecific cells ± dump tetramer exclusion.
CD4+ T-cells from pooled spleen and lymph node from unprimed adult and old mice after tetramer enrichment using anti-His magnetic beads. Shown are representative plots of the CD4+ T-cells binding E641:I-Ab + MCC:I-Ek tetramers pre (left) and post (right) gating out the dump, OVA:I-Ab, tetramer+ cells in (A) adult and (B) old mice. Absolute number of CD44lo (left Y-axis) and CD44hi (right Y-axis) CD4+ T-cells binding E641:I-Ab + MCC:I-Ek tetramers (C) pre and (D) post gating out the dump, OVA:I-Ab, tetramer+ cells. Horizontal bar indicates median (**p < 0.005 ns = non-significant; Mann–Whitney). Δ: Fold change in average numbers from adult to old mice. Data are from three experiments with 4 mice/group.