Paiola M et al. (2023), Larval T Cells Are Functionally Distinct from A...

XB-ART-60370

Immunohorizons 2023 Oct 01;710:696-707. doi: 10.4049/immunohorizons.2300081.

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Larval T Cells Are Functionally Distinct from Adult T Cells in Xenopus laevis.

Paiola M , McGuire CC , Lopez Ruiz V , De Jesús Andino F , Robert J .

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The amphibian Xenopus laevis tadpole provides a unique comparative experimental organism for investigating the roles of innate-like T (iT) cells in tolerogenic immunity during early development. Unlike mammals and adult frogs, where conventional T cells are dominant, tadpoles rely mostly on several prominent distinct subsets of iT cells interacting with cognate nonpolymorphic MHC class I-like molecules. In the present study, to investigate whole T cell responsiveness ontogenesis in X. laevis, we determined in tadpoles and adult frogs the capacity of splenic T cells to proliferate in vivo upon infection with two different pathogens, ranavirus FV3 and Mycobacterium marinum, as well as in vitro upon PHA stimulation using the thymidine analogous 5-ethynyl-2'-deoxyuridine and flow cytometry. We also analyzed by RT-quantitative PCR T cell responsiveness upon PHA stimulation. In vivo tadpole splenic T cells showed limited capacity to proliferate, whereas the in vitro proliferation rate was higher than adult T cells. Gene markers for T cell activation and immediate-early genes induced upon TCR activation were upregulated with similar kinetics in tadpole and adult splenocytes. However, the tadpole T cell signature included a lower amplitude in the TCR signaling, which is a hallmark of mammalian memory-like T cells and iT or "preset" T cells. This study suggests that reminiscent of mammalian neonatal T cells, tadpole T cells are functionally different from their adult counterpart.

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Species referenced: Xenopus laevis
Genes referenced: fos gapdh ifng il4 irf4 kcnh6 myh6 nr4a1 ybx1
GO keywords: cell proliferation [+]

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	FIGURE 1. Proliferation of splenic T cells during FV3 virus infection in X. laevis adults (A) and tadpoles (B). At 3 and 6 d dpi in adult frogs, and at 6 and 9 dpi in tadpoles, EdU incorporation was determined by flow cytometry for splenic CD5+/CD8+ and CD5+/CD8− T cells as well as for CD5+/CD8+ and CD5+/CD8− thymocytes as an internal control. The gating strategy is depicted in Supplemental Fig. 1A. (C, D) Whisker plot from (n = 3–7) of splenic CD5+/CD8+ (C) and CD5+/CD8− T cells (D). (E) FV3 genome copy number determined by qPCR. Statistical significance between groups is denoted by differences in letters (a, b), which was determined using a Kruskal–Wallis test followed by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (p < 0.05). Conditions that are not significantly different have the same letter. Boxes represent 25th and 75th percentiles, and the middle line is the median. Whiskers are the smallest and largest values.
	FIGURE 2. T cell proliferation during Mm infection in X. laevis adults (A) and tadpoles (B). At 6 and 12 dpi in adult frogs and tadpoles, EdU incorporation was determined by flow cytometry for splenic CD5+/CD8+ and CD5+/CD8− T cells. The gating strategy is depicted in Supplemental Fig. 1. (C) Whisker plot (n = 3–7). Statistical significance between groups is denoted by differences in letters (a, b), which was determined using a Kruskal–Wallis test followed by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (p < 0.05). Conditions that are not significantly different have the same letter. Boxes represent 25th and 75th percentiles, and the middle line is the median. Whiskers are the smallest and largest values.
	FIGURE 3. In vitro proliferation of splenic T cells from X. laevis adults (A) and tadpoles (B) stimulated with PHA. EdU incorporation was determined by flow cytometry for splenic CD5+/CD8+ and CD5+/CD8− T cells. (C) Whisker plot (n = 4–7). Statistical significance between groups is denoted by differences in letters (a, b), which was determined using a Kruskal–Wallis test followed by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (p < 0.05). Conditions that are not significantly different have the same letter. Boxes represent 25th and 75th percentiles, and the middle line is the median. Whiskers are the smallest and largest values.
	FIGURE 4. Changes in adult and tadpole splenocyte expression profiles of genes associated with TCR signaling following in vitro PHA stimulation for 2, 6, and 24 h. Data at 24 h of stimulation are represented separately because their relative expression was calculated on the basis of expression of ef1a.L, whereas relative expression at 2 h and 6 h was calculated on the basis of expression of gapdh.S (see text for details). Statistical differences between PBS- and PHA-exposed splenocytes in each condition with Wilcoxon matched-pairs signed-rank test (p < 0.05, **p < 0.01).
	FIGURE 5. Changes in adult and tadpole splenocyte expression profiles of genes encoding X. laevis IL-2 receptors and IL-2 following in vitro PHA stimulation for 2, 6, and 24 h. Data at 24 h of stimulation are represented separately because their relative expression was calculated on the basis of expression of ef1a.L, whereas relative expression at 2 h and 6 h was calculated on the basis of expression of gapdh.S (see text for details). Statistical differences between PBS- and PHA-exposed splenocytes in each condition with Wilcoxon matched-pairs signed-rank test (p < 0.05, **p < 0.01). #Statistical differences between tadpole and adult splenocytes with nonparametric Mann–Whitney U test (##p < 0.01, ###p < 0.001).
	FIGURE 6. Changes in adult and tadpole splenocyte expression profiles of genes encoding X. laevis IL-4/IL-13 and IFN-γ following in vitro PHA stimulation for 2, 6, and 24 h. Data at 24 h of stimulation are represented separately because their relative expression was calculated on the basis of expression of ef1a.L, whereas relative expression at 2 h and 6 h was calculated on the basis of expression of gapdh.S (see text for details). Statistical differences between PBS- and PHA-exposed splenocytes in each condition with Wilcoxon matched-pairs signed-rank test (p < 0.05).
	FIGURE 7. Comparison between tadpoles and adult frogs of nur77.L and irf4.L relative expression levels in splenocytes stimulated with PHA for 6, 12 and 24 h (n = 6–8). Relative expression was calculated using gapdh.S as a reference. #Statistical differences between tadpole and adult splenocytes with nonparametric Mann–Whitney U test (#p < 0.05, ##p < 0.01, ###p < 0.001). Boxes represent 25th and 75th percentiles, and the middle line is the median. Whiskers are the smallest and largest values.
	FIGURE 8. Frequency of CD5+ T cells and CD5 surface signal intensity of tadpole and adult frog splenocytes determined by flow cytometry. Gating strategy is depicted in Supplemental Fig. 1. The graph depicts the frequency of CD5+ T cells in the splenocytes. N = 5. Histograms are representative staining obtained with tadpoles and adult frog splenocytes. Light gray peaks are cells stained with only secondary Ab, serving as controls. Statistical differences between tadpole and adult splenocytes with Wilcoxon matched-pairs signed rank test (*p < 0.01).

References [+] :

Adkins, Murine neonatal lymphocytes show rapid early cell cycle entry and cell division. 2003, Pubmed