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Fig 1
Identification of a unique MHC class I-like interacting iT cell population (iVα45) using reverse genetics. (A) Outbred premetamorphic (stage 54, n = 12) tadpoles were assessed for XNC10 (white) and XNC4 (gray) gene expression. *P < 0.05, **P < 0.005, ***P < 0.0005 statistical difference between indicated groups. Tissues examined are as follows: I, intestine; K, kidney; L, liver; Lu, lung; S, spleen; and T, thymus. Letters at the Top of the bars indicate tissues exhibiting significantly different (P < 0.05) gene expression levels in the thymus for either XNC4 or XNC10 compared with b = S; c = L, d = K, e = I, and f = Lu. (B) Representative RT-PCR analysis of total thymocytes from premetamorphic tadpoles untreated and 1 and 3 d postsublethal gamma-irradiation (10 Gy). (C) Schematic of the shRNA targeting the XNC4 alpha 1 domain. (D–G) Relative gene expression by qPCR of XNC4 (D), XNC10 (E), iVα45-Jα1.14 (F), and iVα6-Jα1.43 (G) in F0 tadpole at developmental stage 52/53 transgenic (Tg) with knockdown (KD) for XNC4 Tg (gray), XNC10 Tg (white), or age-matched dejellied controls (black). Results were normalized to two endodgenous controls (GAPDH and L13) and presented as fold change in expression compared with the lowest level of expression for each individual gene. All results were pools from two independent experiments and presented as individual tadpoles with the mean ± SEM (n = 10–12) with outliers. *P < 0.05, **P < 0.005, ***P < 0.0005 (one-way ANOVA followed by post hoc analysis using Tukey’s multiple comparisons test). (H and I) F0 transgenic or control tadpoles (stage 52/53) were i.p. infected with 1 × 104 pfu FV3 (H) or 300,000 cfu Mm (I), and survival was monitored daily over a 30- and 60-d period, respectively. Each experiment was repeated three times independently (n = 15 in each cohort) and the results were pooled. Survival was determined using Kaplan–Meier, *P < 0.05, **P < 0.005, ***P < 0.0005. Dejellied controls (black squares), XNC4shRNA Tg (gray triangles), and XNC10shRNA Tg (white circles).
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Fig 2
iVα45 T cell loss of function using two different reverse-genetic approaches. (A) Schematic representation showing the modified sgRNA targeting the Jα1.14 gene segment. (B) Bar graph showing the relative contributions of different types of mutations divided into insertions (black), >10-bp deletions (light gray), and <10-bp deletions (dark gray) observed among 10 randomly selected sgRNA/Cas9-treated tadpoles. Note: Multiple alignments of all mutations are shown in SI Appendix, Fig. S6. (C) Expression of the invariant Vα45-Jα1.14 transcript by qPCR in age-matched Cas9-only injected WT controls (black) and CRISPR/Cas9 Jα1.14-injected tadpoles (gray). P = 0.0645 (Student’s t test). (D) Schematic of the shRNA targeting the iVα45-Jα1.14 CDR3 region, dotted line indicates the Vα/Jα junction. Note: No n-nucleotide diversity was observed in any of the iTCR rearrangments. (E–J) Relative gene expression by qPCR in the thymus of stage 52/53 tadpoles transgenic with iVα45-Jα1.14 CDR3 KD (gray) and age-matched dejellied controls (black) for iVα45-Jα1.14 (E), iVα6-Jα1.43 (F), iVα40-Jα1.22 (G), iVα41-Jα1.40 (H), iVα22-Jα1.32 (I), and iVα23-Jα1.3 (J). Results were normalized to two endogenous controls (GAPDH and L13) and presented as fold change in expression compared with the lowest level of expression for each individual gene. All results are pools from two separate experiments and presented as individual tadpoles with the mean ± SE (n = 10–12). **P < 0.005 (one-way ANOVA test followed by a post hoc analysis using Tukey’s multiple comparisons test).
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Fig 3
iVα45 T cells are required for host resistance to Mm but not FV3 infection. F0 transgenic iVα45-Jα1.14 CDR3 KD and control tadpoles (stage 52/53) were sham infected (controls) or either i.p. infected with 1 à 104 pfu FV3 (A and C) or 300,000 cfu Mm (B and D), and survival was monitored daily over a 30- and 60-d period, respectively. Each experiment was repeated three times independently (n = 15 in each cohort) and the results were pooled. Survival was determined using KaplanâMeier, *P < 0.05, ***P < 0.0005. Shown are dejellied controls (black squares), iVα45-Jα1.14 CDR3shRNA Tg (inverted gray triangles), and Jα1.14sgRNA/CRISPR Tg (gray diamonds).
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Fig 4
iVα45-Jα1.14 CDR3shRNA KD specifically ablates iVα45 T cells. Quantitative gene expression analysis of iVα45-Jα1.14, iVα6-Jα143, CD3ε, CD4, CD8β, and XNC4 in liver isolated from either uninfected (unif.) or Mm (300,000 cfu) infected F0 transgenic tadpoles KD for Vα45Jα1.14 CDR3 (gray triangles, n = 10) or age-matched dejellied control (black circles, n = 10) at 0 and 6 dpi. Gene expression was determined relative to the endogenous control GAPDH and normalized against respective uninfected gene expression. Results are representative of two independent experiments and presented with the mean ± SEM (n = 10–12). *P < 0.05, **P < 0.005, (one-way ANOVA test followed by a post hoc analysis using Tukey’s multiple comparisons test). ns, nonsignificant.
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Fig 5
iVα45 T cell deficiency impacts cytokine expression profile in liver of Mm-infected tadpoles. Relative gene expression analysis by qPCR for inducible nitric oxide (iNOS), Arginase 1 (Arg1), interleukin 10 (IL-10), transforming growth factor β (TGFβ), interleukin 1β (IL1β), interleukin 12 (IL12-B), interleukin 18 (IL-18), tumor necrosis factor α (TNFα), and interferon gamma (IFN-γ) in liver isolated from either uninfected or Mm (300,000 cfu)-infected F0 transgenic tadpoles with KD of Vα45Jα1.14 CDR3 (gray triangles, n = 10) or age-matched dejellied control (black circles, n = 10) at 0 and 6 dpi. Gene expression was determined relative to the endogenous control GAPDH and normalized against respective uninfected gene expression. Results are representative of two independent experiments and presented with the mean ± SEM (n = 10–12). *P < 0.05, **P < 0.005, ***P < 0.0005 (one-way ANOVA test followed by a post hoc analysis using Tukey’s multiple comparisons test). ns, nonsignificant.
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