Banach M et al. (2019), Impacts of the MHC class I-like XNC10 and innat...

XB-ART-56003

Carcinogenesis 2019 Jul 20;407:924-935. doi: 10.1093/carcin/bgz100.

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Impacts of the MHC class I-like XNC10 and innate-like T cells on tumor tolerance and rejection in the amphibian Xenopus.

Banach M , Edholm ES , Gonzalez X , Benraiss A , Robert J .

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The conditions that lead to antitumor or protumor functions of natural killer T (NKT) cells against mammalian tumors are only partially understood. Therefore, insights into the evolutionary conservation of NKT and their analogs-innate-like T (iT) cells-may reveal factors that contribute to tumor eradication. As such, we investigated the amphibian Xenopus laevis iT cells and interacting MHC class I-like (XNC or mhc1b.L) genes against ff-2 thymic lymphoid tumors. Upon ff-2 intraperitoneal transplantation into syngeneic tadpoles, two iT cell subsets iVα6 and iVα22, characterized by an invariant T-cell receptor α chain rearrangement (Vα6-Jα1.43 and Vα22-Jα1.32 respectively), were recruited to the peritoneum, concomitant with a decreased level of these transcripts in the spleen and thymus. To address the hypothesize that different iT cell subsets have distinct, possibly opposing, roles upon ff-2 tumor challenge, we determined whether ff-2 tumor growth could be manipulated by impairing Vα6 iT cells or by deleting their restricting element, the XNC gene, XNC10 (mhc1b10.1.L), on ff-2 tumors. Accordingly, the in vivo depletion of Vα6 iT cells using XNC10-tetramers enhanced tumor growth, indicating Vα6 iT cell-mediated antitumor activities. However, XNC10-deficient transgenic tadpoles that also lack Vα6 iT cells were resistant to ff-2 tumors, uncovering a potential new function of XNC10 besides Vα6 iT cell development. Furthermore, the CRISPR/Cas9-mediated knockout of XNC10 in ff-2 tumors broke the immune tolerance. Together, our findings demonstrate the relevance of XNC10/iT cell axis in controlling Xenopus tumor tolerance or rejection.

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Species referenced: Xenopus laevis
Genes referenced: arg1 gapdh mhc1-uba1 myh4 myh6 slc22a6 tnfrsf10b

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	Figure 1. Kinetics of Vα6 iT cells after ff-2 tumor transplantation. Tadpoles at the developmental stage 54–55 were challenged ip with 1 × 105 ff-2 tumor cells. Peritoneal lavages, spleen, thymus and liver were collected from un-, mock- and tumor-challenged tadpoles to assess kinetics of Vα6 iT cells. Mock challenge was performed by ip injection of APBS without tumor cells. (A, B) Tumor cells were additionally stained with PKH26 Cell Tracer (Sigma) before transplantation to discriminate from CD8+ T cells. (A) Gating strategy for flow cytometry analysis (top panels), and cytograms showing 3 days following tumor transplantation in PKH26 gate (middle panels) and non-PKH26 gate (bottom panels) obtained by pooling five peritoneal lavages from tumor-transplanted tadpoles. (B) Number of XNC10-T+ cells in non-PKH26 gate. (C–F) Relative transcript levels of Vα6-Jα1.43 iTCR after ff-2 tumor challenge was quantified in peritoneal cavity (C), spleen (D), thymus (E) and liver (F). Each dot represents one tadpole. Dotted black line indicates the limit of qPCR detection. Gene expression was normalized against GAPDH transcript expression and represented as fold change compared with the lowest level of expression. Results are pooled from three independent experiments and presented as the mean ± SEM (n = 5–17). One-way ANOVA, followed by post hoc Tukey’s multiple comparisons tests were used to determine the significance of differences among groups and defined as P < 0.05, P < 0.005, **P < 0.0005. ns, no statistical significance.
	Figure 1. Kinetics of Vα6 iT cells after ff-2 tumor transplantation. Tadpoles at the developmental stage 54–55 were challenged ip with 1 × 105 ff-2 tumor cells. Peritoneal lavages, spleen, thymus and liver were collected from un-, mock- and tumor-challenged tadpoles to assess kinetics of Vα6 iT cells. Mock challenge was performed by ip injection of APBS without tumor cells. (A, B) Tumor cells were additionally stained with PKH26 Cell Tracer (Sigma) before transplantation to discriminate from CD8+ T cells. (A) Gating strategy for flow cytometry analysis (top panels), and cytograms showing 3 days following tumor transplantation in PKH26 gate (middle panels) and non-PKH26 gate (bottom panels) obtained by pooling five peritoneal lavages from tumor-transplanted tadpoles. (B) Number of XNC10-T+ cells in non-PKH26 gate. (C–F) Relative transcript levels of Vα6-Jα1.43 iTCR after ff-2 tumor challenge was quantified in peritoneal cavity (C), spleen (D), thymus (E) and liver (F). Each dot represents one tadpole. Dotted black line indicates the limit of qPCR detection. Gene expression was normalized against GAPDH transcript expression and represented as fold change compared with the lowest level of expression. Results are pooled from three independent experiments and presented as the mean ± SEM (n = 5–17). One-way ANOVA, followed by post hoc Tukey’s multiple comparisons tests were used to determine the significance of differences among groups and defined as P < 0.05, P < 0.005, **P < 0.0005. ns, no statistical significance.
	Figure 2. Changes in Vα22-Jα1.32 iTCR transcripts following ff-2 tumor transplantation. Tadpoles at the developmental stage 54–55 were ip challenged with 1 × 105 ff-2 tumor cells. Peritoneal cells, spleen, thymus and liver were collected from un-, mock- and tumor-challenged tadpoles. Mock challenge was performed by injecting ip APBS only. (A–D) Relative transcript level of Vα22-Jα1.32 iTCR was measured in peritoneal cavity (A), spleen (B), thymus (C) and liver (D) following ff-2 tumor challenge. Gene expression was normalized against GAPDH expression and represented as fold change compared with the lowest expression level. Dotted black line indicates qPCR detection limit. Results are pooled from three independent experiments and presented as the mean ± SEM (n = 6–21). One-way ANOVA followed by post hoc Tukey’s multiple comparisons tests were used to determine the significance of differences among groups and defined as P < 0.05, P < 0.005, ***P < 0.00005. ns, no statistical significance
	Figure 3. Outcome of ff-2 tumor transplantations into XNC10-deficient tadpoles. WT or XNC10 KD Tg F tadpoles at developmental stage 54–55 were ip transplanted with 1 × 105 ff-2 tumor cells. At indicated day after transplantation, peritoneal lavages were performed, and cells were subjected to flow cytometry. Frequency and cell number of ff-2 tumor cells were assessed with CTX staining. (A–C) Flow cytometry analysis at day 7 following tumor transplantation. (A) Representative FSC-A/SSC-A plots showing cells tumor and peritoneal cells (top) and CTX/MHC class II staining (bottom) obtained from WT and XNC10 KD Tg tadpoles. (B) Frequencies and numbers of CTX+ ff-2 tumor cells. (C) Frequencies and numbers of CTX−/MHC class II+ cells. (D) Frequency of retrieved ff-2 tumor cells, stained with CTX, following 3 days after transplantation in WT and XNC10 KD Tg tadpoles. (E) Relative abundance of GCSF-R transcripts at days 1, 2 and 3 after transplantation in WT and XNC10 KD Tg tadpoles. Gene expression was normalized against GAPDH expression and represented as fold change compared with the lowest level of expression. Results were pooled from three independent experiments and presented as the mean ± SEM (n = 10–25). Two-tailed unpaired t test was used to determine significance of differences between groups and defined as P < 0.05, P < 0.005, ***P < 0.00005 . ns, no statistical significance
	Figure 4. XNC10-T impairs Vα6 iT cells and promotes ff-2 tumor growth in vivo. (A) XNC10-T induced Vα6 iT cell death ex vivo. Freshly isolated splenocytes from an adult outbred X. laevis were stained with four different concentrations of APC-conjugated XNC10-T (5.0, 7.5, 10.0 and 12.5 µg/12.5 µl) for 30 and 90 min. Live/dead cell viability dye—propidium iodide (PI) was used to stain dead cells. (B–F) Two doses of 1 µg XNC10-T in 10 µl APBS were ip administered, 1 day before and 1 day after ip ff-2 WT tumor challenge. Peritoneal cells and spleens were collected to assess gene expression by qPCR and tumor cell count (cytospin followed by Giemsa staining). (B) Transcript levels of Vα6-Jα1.43 iTCR were assessed in peritoneal cavity and spleen following XNC10-T administration in ff-2 tumor-bearing tadpoles. (C, D) Transcript levels of perforin and FasL (C) as well as iNOS and Arg1 (D) were detected in peritoneal cavity. (E) ff-2 tumor cells collected by peritoneal lavage and cell numbers were assessed by cytospin and Giemsa stain. Each dot represents one tadpole. Dotted black line indicates qPCR detection limit. Gene expression was normalized against GAPDH expression and represented as fold change compared with the lowest level of expression. One-way ANOVA followed by post hoc Tukey’s multiple comparisons tests were used to determine significance of differences among groups and defined as P < 0.05, P < 0.005, **P < 0.0005. ns, no statistical significance.
	Figure 4. XNC10-T impairs Vα6 iT cells and promotes ff-2 tumor growth in vivo. (A) XNC10-T induced Vα6 iT cell death ex vivo. Freshly isolated splenocytes from an adult outbred X. laevis were stained with four different concentrations of APC-conjugated XNC10-T (5.0, 7.5, 10.0 and 12.5 µg/12.5 µl) for 30 and 90 min. Live/dead cell viability dye—propidium iodide (PI) was used to stain dead cells. (B–F) Two doses of 1 µg XNC10-T in 10 µl APBS were ip administered, 1 day before and 1 day after ip ff-2 WT tumor challenge. Peritoneal cells and spleens were collected to assess gene expression by qPCR and tumor cell count (cytospin followed by Giemsa staining). (B) Transcript levels of Vα6-Jα1.43 iTCR were assessed in peritoneal cavity and spleen following XNC10-T administration in ff-2 tumor-bearing tadpoles. (C, D) Transcript levels of perforin and FasL (C) as well as iNOS and Arg1 (D) were detected in peritoneal cavity. (E) ff-2 tumor cells collected by peritoneal lavage and cell numbers were assessed by cytospin and Giemsa stain. Each dot represents one tadpole. Dotted black line indicates qPCR detection limit. Gene expression was normalized against GAPDH expression and represented as fold change compared with the lowest level of expression. One-way ANOVA followed by post hoc Tukey’s multiple comparisons tests were used to determine significance of differences among groups and defined as P < 0.05, P < 0.005, **P < 0.0005. ns, no statistical significance.
	Figure 5. Generation and validation of ff-2 XNC10 KO tumor cells. ff-2 cells were transfected via 4D-Nucleofector™ system (Lonza) with two plasmids: sgRNA-XNC10- mCherry-Cas9 and homology template of XNC10 α2 with tRFP-puro cassette. Puromycin-containing media facilitated selection. (A) Transfection efficiency monitored with flow cytometry. (B) Integration of artificial cassette in XNC10 α2 domain, detected by conventional PCR. (C–E) Single cell clones, generated by serial dilution, were screened for the absence of genomic XNC10 PCR product (C), relative expression of XNC10 and other XNC genes (D) and cell surface expression of XNC10, detected by polyclonal α-XNC10 Ab (E). Gene expression was normalized against GAPDH and represented as fold change compared with the lowest level of expression. MFI, median fluorescence intensity: ff-2 WT—61,315; XNC10+/+ clone #3—45,048; XNC10+/− KO clone #9—38,943; XNC10−/− KO clone #5—38,358.
	Figure 6. XNC10 KO ff-2 tumor cells have no proliferative in vitro defect but are rejected by syngeneic F tadpoles. (A) In vitro proliferation rate of XNC10 KO ff-2 tumor cells, assessed by plating 500 000 cells per well of each tumor clone in triplicates. Cell number was evaluated at 24, 48 and 72 h using a hemocytometer and Trepan blue exclusion staining; percentage of dead cells never reached 5% per well. Values are presented as the mean ± SEM. Statistical differences were determined using a nonparametric Mann–Whitney U test. (B and C) Rejection of XNC10 KO ff-2 tumor clones by syngeneic F tadpoles. F tadpoles at the developmental stage 54/55 were ip transplanted with 1 × 105 ff-2 tumor cells: WT, non-mutated XNC10+/+ clone #3, heterozygous XNC10+/− KO clone #9, homozygous XNC10−/− KO clone #5. At day 7 after tumor transplantation, peritoneal lavages were performed, cells counted and stained with CTX for flow cytometry. (B) Representative flow plots of peritoneal exudates stained with CTX mAb. (C) Frequency and cell number of CTX+ ff-2 tumor cells. Data pooled from two independent experiments and represented with the mean ± SEM (n = 3–5). One-way ANOVA followed by post hoc Tukey’s multiple comparisons tests were used to determine the significance of differences among groups and defined as P < 0.01, *P < 0.005. ns, no statistical significance among any paired groups.
	Supplementary Figure 1. iTCR rearrangements that are not altered after ff-2 tumor transplantation. Relative expression of four iTCR α chains: Vα23-Jα1.3, Vα45-Jα1.14, Vα40- Jα1.22, Vα41-Jα1.40 was monitored in the peritoneal cavity, spleen, thymus, and liver after challenge with 1x105 ff-2 WT tumor cells. Tadpoles at the developmental stage 54-55 were used. Peritoneal lavages, spleen, thymus, and liver were collected from un-, mock- and tumor-challenged tadpoles to assess gene expression by qPCR. Mock challenge was performed by injecting APBS only. Each dot represents one tadpole. Dotted black line indicates the limit of qPCR detection. Gene expression was normalized against endogenous GAPDH expression and represented as fold change compared to the lowest level of expression. Results are pooled from three independent experiments and presented as the mean ± SEM (n = 3 – 15). One-way ANOVA followed by post hoc Tukey’s multiple comparisons tests were used to determine significance of differences among groups. ns, no statistical significance among any paired groups.
	Supplementary Figure 2. Relative expression of Vα6-Jα1.43 and Vα22-Jα1.32 iTCR rearrangements in different tissues of unchallenged F tadpoles. Dotted line indicates the qPCR detection limit. Gene expression was normalized against GAPDH expression and represented as fold change compared to the lowest level of expression. Results are pooled from three independent experiments and presented as the mean ± SEM (n = 6 – 21). One-way ANOVA followed by post hoc Tukey’s multiple comparisons tests were used to determine significance of differences among groups and defined as * p < 0.05, *** p < 0.0005.
	Supplementary Figure 3. Unchallenged XNC10 KD F transgenic tadpoles have deficiency in XNC10 and Vα6-Jα1.43 iTCR transcript levels. Spleens of XNC10 deficient (KD) Tg inbred F and WT inbred F control tadpoles at the developmental stage 54-55 were used to assess transcript levels of XNC10 (A), XNC1 (B), Vα6-Jα1.43 (C), and Vα22-Jα1.32 (D) by qPCR. (E) Correlation of XNC10 silencing with reduced Vα6-Jα1.43 gene expression per individual tadpoles, assessed in spleens of unchallenged XNC10 KD inbred F tadpoles. Gene expression was normalized against GAPDH expression and represented as fold change compared to the lowest level of expression. Results were pooled from two independent experiments and presented as the mean ± SEM (n = 5 – 15). Two-tailed unpaired t test was used to determine significance of differences between groups and defined as p < 0.005, * p < 0.0005. ns, no statistical significance.
	Supplementary Figure 4. No differences between WT or XNC10 KD inbred F hosts in relative abundance of MSCF-R transcripts after ff-2 tumor transplantations. WT or XNC10 KD Tg inbred F tadpoles at the developmental stage 54-55 were ip transplanted with 1x105 ff-2 WT tumor cells. Following tumor transplantation, peritoneal lavages were collected to assess gene expression by qPCR. Gene expression was normalized against GAPDH expression and represented as fold change compared to the lowest level of expression. Results were pooled from three independent experiments and presented as the mean ± SEM (n = 9 – 20). Two-tailed unpaired t test was used to determine significance of differences between groups. ns, no statistical significance.
	Supplementary Figure 5. Effects of XNC10-Tetramer treatment. (A) XNC10-T induced Vα6 iT cell death ex vivo represented as cell numbers. Freshly isolated splenocytes from an outbred X. laevis adult were stained with 4 different concentrations of APC-conjugated XNC10-T (5.0, 7.5, 10.0 and 12.5 µg/12.5 µl) for 30 min and 90 min. (B) XNC10-T did not affect the viability of cells other than Vα6 iT cells ex vivo. Freshly isolated splenocytes from an adult outbred X. laevis were stained with 4 different concentrations of APC-conjugated XNC10-T (5.0, 7.5, 10.0 and 12.5 µg/12.5 µl) for 30 min and 90 min. Live/dead cell viability dye – propidium iodide (PI) was used to stain dead cells. (C) Enhanced XNC10-T induced Vα6 iT cell death ex vivo at room temperature (RT).
	Supplementary Figure 6. Changes in Vα22-Jα1.32 iTCR transcript levels following ff-2 tumor transplantation and XNC10-T administration. Tadpoles at the developmental stage 54-55 were used and peritoneal cells were collected for transcriptional analysis. Each dot represents one tadpole. Dotted line indicates the qPCR detection limit. Gene expression was normalized against GAPDH expression and represented as fold change compared to the lowest level of expression. Results are presented as the mean ± SEM (n = 4 – 7). One-way ANOVA followed by post hoc Tukey’s multiple comparisons tests were used to determine significance of differences among groups and defined as * p < 0.05. ns, no statistical significance.
	Supplementary Figure 7. Working model of interactions between Xenopus ff-2 lymphoid tumor and two different iT cell subsets in the peritoneal cavity of tumor-challenged tadpoles. Upon intraperitoneal tumor transplantation, both Vα6 iT and Vα22 iT cells infiltrate into the peritoneal cavity. Vα6 iT cells either directly or indirectly limit tumor growth. Expression of XNC10 on tumor cells suppresses Vα6 iT cell cytotoxicity, promoting tumor growth. The ligand for Vα22 iT cells is unknown. By impairing Vα6 iT cells or tumor expression of XNC10 genes, tumor growth can be manipulated. Accordingly, XNC10 deficient ff-2 tumor cells are rejected by WT inbred F tadpoles. Alternatively, blocking Vα6 iT cell function with XNC10-tetramer enhances ff-2 WT tumor growth. Surprisingly, F tadpoles with XNC10 deficiency reject transplanted ff-2 tumors.

References [+] :

Banach, Tumor immunology viewed from alternative animal models-the Xenopus story. 2017, Pubmed, Xenbase

	Figure 1. Kinetics of Vα6 iT cells after ff-2 tumor transplantation. Tadpoles at the developmental stage 54–55 were challenged ip with 1 × 105 ff-2 tumor cells. Peritoneal lavages, spleen, thymus and liver were collected from un-, mock- and tumor-challenged tadpoles to assess kinetics of Vα6 iT cells. Mock challenge was performed by ip injection of APBS without tumor cells. (A, B) Tumor cells were additionally stained with PKH26 Cell Tracer (Sigma) before transplantation to discriminate from CD8+ T cells. (A) Gating strategy for flow cytometry analysis (top panels), and cytograms showing 3 days following tumor transplantation in PKH26 gate (middle panels) and non-PKH26 gate (bottom panels) obtained by pooling five peritoneal lavages from tumor-transplanted tadpoles. (B) Number of XNC10-T+ cells in non-PKH26 gate. (C–F) Relative transcript levels of Vα6-Jα1.43 iTCR after ff-2 tumor challenge was quantified in peritoneal cavity (C), spleen (D), thymus (E) and liver (F). Each dot represents one tadpole. Dotted black line indicates the limit of qPCR detection. Gene expression was normalized against GAPDH transcript expression and represented as fold change compared with the lowest level of expression. Results are pooled from three independent experiments and presented as the mean ± SEM (n = 5–17). One-way ANOVA, followed by post hoc Tukey’s multiple comparisons tests were used to determine the significance of differences among groups and defined as P < 0.05, P < 0.005, **P < 0.0005. ns, no statistical significance.
	Figure 1. Kinetics of Vα6 iT cells after ff-2 tumor transplantation. Tadpoles at the developmental stage 54–55 were challenged ip with 1 × 105 ff-2 tumor cells. Peritoneal lavages, spleen, thymus and liver were collected from un-, mock- and tumor-challenged tadpoles to assess kinetics of Vα6 iT cells. Mock challenge was performed by ip injection of APBS without tumor cells. (A, B) Tumor cells were additionally stained with PKH26 Cell Tracer (Sigma) before transplantation to discriminate from CD8+ T cells. (A) Gating strategy for flow cytometry analysis (top panels), and cytograms showing 3 days following tumor transplantation in PKH26 gate (middle panels) and non-PKH26 gate (bottom panels) obtained by pooling five peritoneal lavages from tumor-transplanted tadpoles. (B) Number of XNC10-T+ cells in non-PKH26 gate. (C–F) Relative transcript levels of Vα6-Jα1.43 iTCR after ff-2 tumor challenge was quantified in peritoneal cavity (C), spleen (D), thymus (E) and liver (F). Each dot represents one tadpole. Dotted black line indicates the limit of qPCR detection. Gene expression was normalized against GAPDH transcript expression and represented as fold change compared with the lowest level of expression. Results are pooled from three independent experiments and presented as the mean ± SEM (n = 5–17). One-way ANOVA, followed by post hoc Tukey’s multiple comparisons tests were used to determine the significance of differences among groups and defined as P < 0.05, P < 0.005, **P < 0.0005. ns, no statistical significance.
	Figure 2. Changes in Vα22-Jα1.32 iTCR transcripts following ff-2 tumor transplantation. Tadpoles at the developmental stage 54–55 were ip challenged with 1 × 105 ff-2 tumor cells. Peritoneal cells, spleen, thymus and liver were collected from un-, mock- and tumor-challenged tadpoles. Mock challenge was performed by injecting ip APBS only. (A–D) Relative transcript level of Vα22-Jα1.32 iTCR was measured in peritoneal cavity (A), spleen (B), thymus (C) and liver (D) following ff-2 tumor challenge. Gene expression was normalized against GAPDH expression and represented as fold change compared with the lowest expression level. Dotted black line indicates qPCR detection limit. Results are pooled from three independent experiments and presented as the mean ± SEM (n = 6–21). One-way ANOVA followed by post hoc Tukey’s multiple comparisons tests were used to determine the significance of differences among groups and defined as P < 0.05, P < 0.005, ***P < 0.00005. ns, no statistical significance
	Figure 3. Outcome of ff-2 tumor transplantations into XNC10-deficient tadpoles. WT or XNC10 KD Tg F tadpoles at developmental stage 54–55 were ip transplanted with 1 × 105 ff-2 tumor cells. At indicated day after transplantation, peritoneal lavages were performed, and cells were subjected to flow cytometry. Frequency and cell number of ff-2 tumor cells were assessed with CTX staining. (A–C) Flow cytometry analysis at day 7 following tumor transplantation. (A) Representative FSC-A/SSC-A plots showing cells tumor and peritoneal cells (top) and CTX/MHC class II staining (bottom) obtained from WT and XNC10 KD Tg tadpoles. (B) Frequencies and numbers of CTX+ ff-2 tumor cells. (C) Frequencies and numbers of CTX−/MHC class II+ cells. (D) Frequency of retrieved ff-2 tumor cells, stained with CTX, following 3 days after transplantation in WT and XNC10 KD Tg tadpoles. (E) Relative abundance of GCSF-R transcripts at days 1, 2 and 3 after transplantation in WT and XNC10 KD Tg tadpoles. Gene expression was normalized against GAPDH expression and represented as fold change compared with the lowest level of expression. Results were pooled from three independent experiments and presented as the mean ± SEM (n = 10–25). Two-tailed unpaired t test was used to determine significance of differences between groups and defined as P < 0.05, P < 0.005, ***P < 0.00005 . ns, no statistical significance
	Figure 4. XNC10-T impairs Vα6 iT cells and promotes ff-2 tumor growth in vivo. (A) XNC10-T induced Vα6 iT cell death ex vivo. Freshly isolated splenocytes from an adult outbred X. laevis were stained with four different concentrations of APC-conjugated XNC10-T (5.0, 7.5, 10.0 and 12.5 µg/12.5 µl) for 30 and 90 min. Live/dead cell viability dye—propidium iodide (PI) was used to stain dead cells. (B–F) Two doses of 1 µg XNC10-T in 10 µl APBS were ip administered, 1 day before and 1 day after ip ff-2 WT tumor challenge. Peritoneal cells and spleens were collected to assess gene expression by qPCR and tumor cell count (cytospin followed by Giemsa staining). (B) Transcript levels of Vα6-Jα1.43 iTCR were assessed in peritoneal cavity and spleen following XNC10-T administration in ff-2 tumor-bearing tadpoles. (C, D) Transcript levels of perforin and FasL (C) as well as iNOS and Arg1 (D) were detected in peritoneal cavity. (E) ff-2 tumor cells collected by peritoneal lavage and cell numbers were assessed by cytospin and Giemsa stain. Each dot represents one tadpole. Dotted black line indicates qPCR detection limit. Gene expression was normalized against GAPDH expression and represented as fold change compared with the lowest level of expression. One-way ANOVA followed by post hoc Tukey’s multiple comparisons tests were used to determine significance of differences among groups and defined as P < 0.05, P < 0.005, **P < 0.0005. ns, no statistical significance.
	Figure 4. XNC10-T impairs Vα6 iT cells and promotes ff-2 tumor growth in vivo. (A) XNC10-T induced Vα6 iT cell death ex vivo. Freshly isolated splenocytes from an adult outbred X. laevis were stained with four different concentrations of APC-conjugated XNC10-T (5.0, 7.5, 10.0 and 12.5 µg/12.5 µl) for 30 and 90 min. Live/dead cell viability dye—propidium iodide (PI) was used to stain dead cells. (B–F) Two doses of 1 µg XNC10-T in 10 µl APBS were ip administered, 1 day before and 1 day after ip ff-2 WT tumor challenge. Peritoneal cells and spleens were collected to assess gene expression by qPCR and tumor cell count (cytospin followed by Giemsa staining). (B) Transcript levels of Vα6-Jα1.43 iTCR were assessed in peritoneal cavity and spleen following XNC10-T administration in ff-2 tumor-bearing tadpoles. (C, D) Transcript levels of perforin and FasL (C) as well as iNOS and Arg1 (D) were detected in peritoneal cavity. (E) ff-2 tumor cells collected by peritoneal lavage and cell numbers were assessed by cytospin and Giemsa stain. Each dot represents one tadpole. Dotted black line indicates qPCR detection limit. Gene expression was normalized against GAPDH expression and represented as fold change compared with the lowest level of expression. One-way ANOVA followed by post hoc Tukey’s multiple comparisons tests were used to determine significance of differences among groups and defined as P < 0.05, P < 0.005, **P < 0.0005. ns, no statistical significance.
	Figure 5. Generation and validation of ff-2 XNC10 KO tumor cells. ff-2 cells were transfected via 4D-Nucleofector™ system (Lonza) with two plasmids: sgRNA-XNC10- mCherry-Cas9 and homology template of XNC10 α2 with tRFP-puro cassette. Puromycin-containing media facilitated selection. (A) Transfection efficiency monitored with flow cytometry. (B) Integration of artificial cassette in XNC10 α2 domain, detected by conventional PCR. (C–E) Single cell clones, generated by serial dilution, were screened for the absence of genomic XNC10 PCR product (C), relative expression of XNC10 and other XNC genes (D) and cell surface expression of XNC10, detected by polyclonal α-XNC10 Ab (E). Gene expression was normalized against GAPDH and represented as fold change compared with the lowest level of expression. MFI, median fluorescence intensity: ff-2 WT—61,315; XNC10+/+ clone #3—45,048; XNC10+/− KO clone #9—38,943; XNC10−/− KO clone #5—38,358.
	Figure 6. XNC10 KO ff-2 tumor cells have no proliferative in vitro defect but are rejected by syngeneic F tadpoles. (A) In vitro proliferation rate of XNC10 KO ff-2 tumor cells, assessed by plating 500 000 cells per well of each tumor clone in triplicates. Cell number was evaluated at 24, 48 and 72 h using a hemocytometer and Trepan blue exclusion staining; percentage of dead cells never reached 5% per well. Values are presented as the mean ± SEM. Statistical differences were determined using a nonparametric Mann–Whitney U test. (B and C) Rejection of XNC10 KO ff-2 tumor clones by syngeneic F tadpoles. F tadpoles at the developmental stage 54/55 were ip transplanted with 1 × 105 ff-2 tumor cells: WT, non-mutated XNC10+/+ clone #3, heterozygous XNC10+/− KO clone #9, homozygous XNC10−/− KO clone #5. At day 7 after tumor transplantation, peritoneal lavages were performed, cells counted and stained with CTX for flow cytometry. (B) Representative flow plots of peritoneal exudates stained with CTX mAb. (C) Frequency and cell number of CTX+ ff-2 tumor cells. Data pooled from two independent experiments and represented with the mean ± SEM (n = 3–5). One-way ANOVA followed by post hoc Tukey’s multiple comparisons tests were used to determine the significance of differences among groups and defined as P < 0.01, *P < 0.005. ns, no statistical significance among any paired groups.
	Supplementary Figure 1. iTCR rearrangements that are not altered after ff-2 tumor transplantation. Relative expression of four iTCR α chains: Vα23-Jα1.3, Vα45-Jα1.14, Vα40- Jα1.22, Vα41-Jα1.40 was monitored in the peritoneal cavity, spleen, thymus, and liver after challenge with 1x105 ff-2 WT tumor cells. Tadpoles at the developmental stage 54-55 were used. Peritoneal lavages, spleen, thymus, and liver were collected from un-, mock- and tumor-challenged tadpoles to assess gene expression by qPCR. Mock challenge was performed by injecting APBS only. Each dot represents one tadpole. Dotted black line indicates the limit of qPCR detection. Gene expression was normalized against endogenous GAPDH expression and represented as fold change compared to the lowest level of expression. Results are pooled from three independent experiments and presented as the mean ± SEM (n = 3 – 15). One-way ANOVA followed by post hoc Tukey’s multiple comparisons tests were used to determine significance of differences among groups. ns, no statistical significance among any paired groups.
	Supplementary Figure 2. Relative expression of Vα6-Jα1.43 and Vα22-Jα1.32 iTCR rearrangements in different tissues of unchallenged F tadpoles. Dotted line indicates the qPCR detection limit. Gene expression was normalized against GAPDH expression and represented as fold change compared to the lowest level of expression. Results are pooled from three independent experiments and presented as the mean ± SEM (n = 6 – 21). One-way ANOVA followed by post hoc Tukey’s multiple comparisons tests were used to determine significance of differences among groups and defined as * p < 0.05, *** p < 0.0005.
	Supplementary Figure 3. Unchallenged XNC10 KD F transgenic tadpoles have deficiency in XNC10 and Vα6-Jα1.43 iTCR transcript levels. Spleens of XNC10 deficient (KD) Tg inbred F and WT inbred F control tadpoles at the developmental stage 54-55 were used to assess transcript levels of XNC10 (A), XNC1 (B), Vα6-Jα1.43 (C), and Vα22-Jα1.32 (D) by qPCR. (E) Correlation of XNC10 silencing with reduced Vα6-Jα1.43 gene expression per individual tadpoles, assessed in spleens of unchallenged XNC10 KD inbred F tadpoles. Gene expression was normalized against GAPDH expression and represented as fold change compared to the lowest level of expression. Results were pooled from two independent experiments and presented as the mean ± SEM (n = 5 – 15). Two-tailed unpaired t test was used to determine significance of differences between groups and defined as p < 0.005, * p < 0.0005. ns, no statistical significance.
	Supplementary Figure 4. No differences between WT or XNC10 KD inbred F hosts in relative abundance of MSCF-R transcripts after ff-2 tumor transplantations. WT or XNC10 KD Tg inbred F tadpoles at the developmental stage 54-55 were ip transplanted with 1x105 ff-2 WT tumor cells. Following tumor transplantation, peritoneal lavages were collected to assess gene expression by qPCR. Gene expression was normalized against GAPDH expression and represented as fold change compared to the lowest level of expression. Results were pooled from three independent experiments and presented as the mean ± SEM (n = 9 – 20). Two-tailed unpaired t test was used to determine significance of differences between groups. ns, no statistical significance.
	Supplementary Figure 5. Effects of XNC10-Tetramer treatment. (A) XNC10-T induced Vα6 iT cell death ex vivo represented as cell numbers. Freshly isolated splenocytes from an outbred X. laevis adult were stained with 4 different concentrations of APC-conjugated XNC10-T (5.0, 7.5, 10.0 and 12.5 µg/12.5 µl) for 30 min and 90 min. (B) XNC10-T did not affect the viability of cells other than Vα6 iT cells ex vivo. Freshly isolated splenocytes from an adult outbred X. laevis were stained with 4 different concentrations of APC-conjugated XNC10-T (5.0, 7.5, 10.0 and 12.5 µg/12.5 µl) for 30 min and 90 min. Live/dead cell viability dye – propidium iodide (PI) was used to stain dead cells. (C) Enhanced XNC10-T induced Vα6 iT cell death ex vivo at room temperature (RT).
	Supplementary Figure 6. Changes in Vα22-Jα1.32 iTCR transcript levels following ff-2 tumor transplantation and XNC10-T administration. Tadpoles at the developmental stage 54-55 were used and peritoneal cells were collected for transcriptional analysis. Each dot represents one tadpole. Dotted line indicates the qPCR detection limit. Gene expression was normalized against GAPDH expression and represented as fold change compared to the lowest level of expression. Results are presented as the mean ± SEM (n = 4 – 7). One-way ANOVA followed by post hoc Tukey’s multiple comparisons tests were used to determine significance of differences among groups and defined as * p < 0.05. ns, no statistical significance.
	Supplementary Figure 7. Working model of interactions between Xenopus ff-2 lymphoid tumor and two different iT cell subsets in the peritoneal cavity of tumor-challenged tadpoles. Upon intraperitoneal tumor transplantation, both Vα6 iT and Vα22 iT cells infiltrate into the peritoneal cavity. Vα6 iT cells either directly or indirectly limit tumor growth. Expression of XNC10 on tumor cells suppresses Vα6 iT cell cytotoxicity, promoting tumor growth. The ligand for Vα22 iT cells is unknown. By impairing Vα6 iT cells or tumor expression of XNC10 genes, tumor growth can be manipulated. Accordingly, XNC10 deficient ff-2 tumor cells are rejected by WT inbred F tadpoles. Alternatively, blocking Vα6 iT cell function with XNC10-tetramer enhances ff-2 WT tumor growth. Surprisingly, F tadpoles with XNC10 deficiency reject transplanted ff-2 tumors.