γδ T cells are abundant T cell population at the mucosa and are important in providing immune surveillance as well as maintaining tissue homeostasis. However, despite γδ T cells origin in thymus, detailed mechanisms regulating γδ T cell development remain poorly understood. *N*6-methyladenosine (m6A) represents one of the most common post-transcriptional modifications of mRNA in mammalian cells, but whether it plays a role in γδ T cell biology is still unclear. Here we show that depletion of m6A demethylase ALKBH5 in lymphocytes specifically induces an expansion of γδ T cells, which confers enhanced protection against gastrointestinal *S. typhimurium* infection. Mechanistically, loss of ALKBH5 favors the development of γδ T cell precursors by increasing the abundance of m6A RNA modification in thymocytes, which further reduces the expression of several target genes including Notch signaling components *Jagged1* and *Notch2*. As a result, impairment of Jagged1/Notch2 signaling contributes to enhanced proliferation and differentiation of γδ T cell precursors, leading to an expanded mature γδ T cell repertoire. Taken together, our results indicate a checkpoint role of ALKBH5 and m6A modification in the regulation of γδ T cell early development.

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There is **NO** Competing Interest.

This is a list of supplementary files associated with this preprint. Click to download.

- ExtendedDataFigure1.tif
Extended Data Fig. 1 ALKBH5 is expressed in a wide range of immune cells. a, Normalized expression value of ALKBH5 across different immune cells. Data is acquired from Immunological Genome Project (www.immgen.org). b, Schematic representation of the targeting strategy used to flank exon 1 of the Alkbh5 loci with loxP sites (top) and excision with Cre-recombinase (bottom). c, Mouse models employed to specifically knock-out ALKBH5 in lymphocyte populations. d, Quantitative PCR (qPCR) analysis of ALKBH5 gene expression level in thymocytes isolated from 4 pairs of Alkbh5f/f Lck+ and Alkbh5f/f cohoused mice (n = 4). Data represent mean ± s.d.; paired t test was used for statistical analysis. **P < 0.01.

- ExtendedDataFigure1.tif
Extended Data Fig. 1 ALKBH5 is expressed in a wide range of immune cells. a, Normalized expression value of ALKBH5 across different immune cells. Data is acquired from Immunological Genome Project (www.immgen.org). b, Schematic representation of the targeting strategy used to flank exon 1 of the Alkbh5 loci with loxP sites (top) and excision with Cre-recombinase (bottom). c, Mouse models employed to specifically knock-out ALKBH5 in lymphocyte populations. d, Quantitative PCR (qPCR) analysis of ALKBH5 gene expression level in thymocytes isolated from 4 pairs of Alkbh5f/f Lck+ and Alkbh5f/f cohoused mice (n = 4). Data represent mean ± s.d.; paired t test was used for statistical analysis. **P < 0.01.

- ExtendedDataFigure2.tif
Extended Data Fig. 2 ALKBH5 deficiency leads to expanded γδ T cell population in the periphery. a, Flow cytometric analysis of lymphocytes isolated from indicated lymphoid organs and tissues (pLN, peripheral lymph node; mLN, mesenteric lymph node) of Alkbh5f/f Lck+ and Alkbh5f/f mice. b, Statistical analysis of cell numbers in (a) is reported. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 7; KO, n = 6). Data represent one out of three independent experiments (mean ± s.d.). **P < 0.01, ***P < 0.001, n.s., not significant.

- ExtendedDataFigure2.tif
Extended Data Fig. 2 ALKBH5 deficiency leads to expanded γδ T cell population in the periphery. a, Flow cytometric analysis of lymphocytes isolated from indicated lymphoid organs and tissues (pLN, peripheral lymph node; mLN, mesenteric lymph node) of Alkbh5f/f Lck+ and Alkbh5f/f mice. b, Statistical analysis of cell numbers in (a) is reported. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 7; KO, n = 6). Data represent one out of three independent experiments (mean ± s.d.). **P < 0.01, ***P < 0.001, n.s., not significant.

- ExtendedDataFigure3.tif
Extended Data Fig. 3 Loss of ALKBH5 has minimum effects on αβ T cell development. a,c,e, Flow cytometric analysis of CD4+ and CD8+ T cells isolated from the spleen (a), pLN (c) and mLN (e) of Alkbh5f/f Lck+ and Alkbh5f/f mice. The right panel reports the statistical analysis of frequencies for CD4+ and CD8+ T cells on the left. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 7; KO, n = 6). Data represent one out of three independent experiments (mean ± s.d.). b,d,f, Flow cytometric analysis of naïve and memory T cells isolated from the spleen (b), pLN (d) and mLN (f) of Alkbh5f/f Lck+ and Alkbh5f/f mice. The right panel reports the statistical analysis of frequencies for effector memory (CD44), naïve (CD62L), central memory (DP) and double-negative (DN) cells on the left. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 7; KO, n = 6). Data represent one out of three independent experiments (mean ± s.d.). *P < 0.05, **P < 0.01, n.s., not significant.

- ExtendedDataFigure3.tif
Extended Data Fig. 3 Loss of ALKBH5 has minimum effects on αβ T cell development. a,c,e, Flow cytometric analysis of CD4+ and CD8+ T cells isolated from the spleen (a), pLN (c) and mLN (e) of Alkbh5f/f Lck+ and Alkbh5f/f mice. The right panel reports the statistical analysis of frequencies for CD4+ and CD8+ T cells on the left. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 7; KO, n = 6). Data represent one out of three independent experiments (mean ± s.d.). b,d,f, Flow cytometric analysis of naïve and memory T cells isolated from the spleen (b), pLN (d) and mLN (f) of Alkbh5f/f Lck+ and Alkbh5f/f mice. The right panel reports the statistical analysis of frequencies for effector memory (CD44), naïve (CD62L), central memory (DP) and double-negative (DN) cells on the left. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 7; KO, n = 6). Data represent one out of three independent experiments (mean ± s.d.). *P < 0.05, **P < 0.01, n.s., not significant.

- ExtendedDataFigure4.tif
Extended Data Fig. 4 Loss of ALKBH5 has minimum effects on αβ T cell development (continued). a,c,e, Statistical analysis of cell numbers for CD4+ and CD8+ T cells isolated from the spleen (a), pLN (c) and mLN (e) of Alkbh5f/f Lck+ and Alkbh5f/f mice is reported, correlated with Extended Data Fig. 3 (a,c,e), respectively. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 7; KO, n = 6). Data represent one out of three independent experiments (mean ± s.d.). b,d,f, Statistical analysis of cell numbers for naïve and memory T cells isolated from the spleen (b), pLN (d) and mLN (f) of Alkbh5f/f Lck+ and Alkbh5f/f is reported, correlated with Extended Data Fig. 3 (b,d,f), respectively. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 7; KO, n = 6). Data represent one out of three independent experiments (mean ± s.d.). *P < 0.05, **P < 0.01, n.s., not significant.

- ExtendedDataFigure4.tif
Extended Data Fig. 4 Loss of ALKBH5 has minimum effects on αβ T cell development (continued). a,c,e, Statistical analysis of cell numbers for CD4+ and CD8+ T cells isolated from the spleen (a), pLN (c) and mLN (e) of Alkbh5f/f Lck+ and Alkbh5f/f mice is reported, correlated with Extended Data Fig. 3 (a,c,e), respectively. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 7; KO, n = 6). Data represent one out of three independent experiments (mean ± s.d.). b,d,f, Statistical analysis of cell numbers for naïve and memory T cells isolated from the spleen (b), pLN (d) and mLN (f) of Alkbh5f/f Lck+ and Alkbh5f/f is reported, correlated with Extended Data Fig. 3 (b,d,f), respectively. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 7; KO, n = 6). Data represent one out of three independent experiments (mean ± s.d.). *P < 0.05, **P < 0.01, n.s., not significant.

- ExtendedDataFigure5.tif
Extended Data Fig. 5 Different subsets of mature γδ T cells are all expanded in Alkbh5f/f Lck+ mice. a, Flow cytometric analysis of three mature γδ T cell subsets (CD8αβ: CD8α+, CD8β+, CD8αα: CD8α+, CD8β–, CD8–: CD8α–, CD8β–) isolated from indicated organs of either Alkbh5f/f Lck+ or Alkbh5f/f mice. b,c, Statistical analysis of frequencies (b) and cell numbers (c) for three mature γδ T cell subsets in (a) is reported. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 7; KO, n = 5). Data represent one out of three independent experiments (mean ± s.d.). **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., not significant.

- ExtendedDataFigure5.tif
Extended Data Fig. 5 Different subsets of mature γδ T cells are all expanded in Alkbh5f/f Lck+ mice. a, Flow cytometric analysis of three mature γδ T cell subsets (CD8αβ: CD8α+, CD8β+, CD8αα: CD8α+, CD8β–, CD8–: CD8α–, CD8β–) isolated from indicated organs of either Alkbh5f/f Lck+ or Alkbh5f/f mice. b,c, Statistical analysis of frequencies (b) and cell numbers (c) for three mature γδ T cell subsets in (a) is reported. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 7; KO, n = 5). Data represent one out of three independent experiments (mean ± s.d.). **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., not significant.

- ExtendedDataFigure6.tif
Extended Data Fig. 6 Loss of ALKBH5 has no impact on IFN-γ or IL-17 production of mature γδ T cells in colon. a, Statistical analysis of frequencies for IFN-γ-/IL-17A-producing mature γδ T cells isolated from Colon-IEL of Alkbh5f/f Lck+ (n = 7) and Alkbh5f/f (n = 6) mice. Unpaired t test was used for statistical analysis. Each dot represents one mouse. Data represent one out of three independent experiments (mean ± s.d.). b, Statistical analysis of frequencies for IFN-γ-/IL-17A-producing mature γδ T cells isolated from Colon-LPL of Alkbh5f/f Lck+ (n = 7) and Alkbh5f/f (n = 6) mice. Unpaired t test was used for statistical analysis. Each dot represents one mouse. Data represent one out of three independent experiments (mean ± s.d.). n.s., not significant.

- ExtendedDataFigure6.tif
Extended Data Fig. 6 Loss of ALKBH5 has no impact on IFN-γ or IL-17 production of mature γδ T cells in colon. a, Statistical analysis of frequencies for IFN-γ-/IL-17A-producing mature γδ T cells isolated from Colon-IEL of Alkbh5f/f Lck+ (n = 7) and Alkbh5f/f (n = 6) mice. Unpaired t test was used for statistical analysis. Each dot represents one mouse. Data represent one out of three independent experiments (mean ± s.d.). b, Statistical analysis of frequencies for IFN-γ-/IL-17A-producing mature γδ T cells isolated from Colon-LPL of Alkbh5f/f Lck+ (n = 7) and Alkbh5f/f (n = 6) mice. Unpaired t test was used for statistical analysis. Each dot represents one mouse. Data represent one out of three independent experiments (mean ± s.d.). n.s., not significant.

- ExtendedDataFigure7.tif
Extended Data Fig. 7 Ratio of thymic γδ T cells subsets and their precursor subsets remain intact or increase slightly in Alkbh5f/f Lck+ mice. a,c,e, Flow cytometric analysis of immature TCRvγ1.1+ γδ T cells at double negative (DN) stage (a), TCRvγ1.1+ γδ T precursor cells (defined as CD73+ TCRvγ1.1+ γδ T cells at DN stage) (c) and NKTvγ1.1 precursor cells (defined as CD73+ CD24– NK1.1+ TCRvγ1.1+ γδ T precursor cells) (e). The right panels report the statistical analysis of frequencies for the populations gated in the left panels. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 7; KO, n = 6). Data represent one out of three independent experiments (mean ± s.d.). b,d,f, Flow cytometric analysis of immature TCRvγ2+ γδ T cells at DN stage (b), TCRvγ2+ γδ T precursor cells (defined as CD73+ TCRvγ2+ γδ T cells at DN stage) (d) and NKTvγ2 precursor cells (defined as CD73+ CD24– NK1.1+ TCRvγ2+ γδ T precursor cells) (f). The right panels report the statistical analysis of frequencies for the populations gated in the left panels. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 7; KO, n = 6). Data represent one out of three independent experiments (mean ± s.d.). *P < 0.05, n.s., not significant.

- ExtendedDataFigure7.tif
Extended Data Fig. 7 Ratio of thymic γδ T cells subsets and their precursor subsets remain intact or increase slightly in Alkbh5f/f Lck+ mice. a,c,e, Flow cytometric analysis of immature TCRvγ1.1+ γδ T cells at double negative (DN) stage (a), TCRvγ1.1+ γδ T precursor cells (defined as CD73+ TCRvγ1.1+ γδ T cells at DN stage) (c) and NKTvγ1.1 precursor cells (defined as CD73+ CD24– NK1.1+ TCRvγ1.1+ γδ T precursor cells) (e). The right panels report the statistical analysis of frequencies for the populations gated in the left panels. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 7; KO, n = 6). Data represent one out of three independent experiments (mean ± s.d.). b,d,f, Flow cytometric analysis of immature TCRvγ2+ γδ T cells at DN stage (b), TCRvγ2+ γδ T precursor cells (defined as CD73+ TCRvγ2+ γδ T cells at DN stage) (d) and NKTvγ2 precursor cells (defined as CD73+ CD24– NK1.1+ TCRvγ2+ γδ T precursor cells) (f). The right panels report the statistical analysis of frequencies for the populations gated in the left panels. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 7; KO, n = 6). Data represent one out of three independent experiments (mean ± s.d.). *P < 0.05, n.s., not significant.

- ExtendedDataFigure8.tif
Extended Data Fig. 8 All subsets of thymic mature γδ T cells are expanded in Alkbh5f/f Lck+ mice based on TCRvγ. a, Flow cytometric analysis of thymic mature TCRvγ1.1+ γδ T cells isolated from Alkbh5f/f Lck+ and Alkbh5f/f mice. b,c, Statistical analysis of frequency (b) and cell number (c) for the gated population in (a) is reported. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 6; KO, n = 5). Data represent one out of three independent experiments (mean ± s.d.). d, Flow cytometric analysis of thymic mature TCRvγ2+ γδ T cells isolated from Alkbh5f/f Lck+ and Alkbh5f/f mice. e,f, Statistical analysis of frequency (e) and cell number (f) for the gated population in (d) is reported. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 6; KO, n = 5). Data represent one out of three independent experiments (mean ± s.d.). g, Flow cytometric analysis of thymic mature γδNKT cells isolated from Alkbh5f/f Lck+ and Alkbh5f/f mice. h,i, Statistical analysis of frequency (h) and cell number (i) for the gated population in (g) is reported. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 6; KO, n = 5). Data represent one out of three independent experiments (mean ± s.d.). ***P < 0.001, ****P < 0.0001, n.s., not significant.

- ExtendedDataFigure8.tif
Extended Data Fig. 8 All subsets of thymic mature γδ T cells are expanded in Alkbh5f/f Lck+ mice based on TCRvγ. a, Flow cytometric analysis of thymic mature TCRvγ1.1+ γδ T cells isolated from Alkbh5f/f Lck+ and Alkbh5f/f mice. b,c, Statistical analysis of frequency (b) and cell number (c) for the gated population in (a) is reported. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 6; KO, n = 5). Data represent one out of three independent experiments (mean ± s.d.). d, Flow cytometric analysis of thymic mature TCRvγ2+ γδ T cells isolated from Alkbh5f/f Lck+ and Alkbh5f/f mice. e,f, Statistical analysis of frequency (e) and cell number (f) for the gated population in (d) is reported. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 6; KO, n = 5). Data represent one out of three independent experiments (mean ± s.d.). g, Flow cytometric analysis of thymic mature γδNKT cells isolated from Alkbh5f/f Lck+ and Alkbh5f/f mice. h,i, Statistical analysis of frequency (h) and cell number (i) for the gated population in (g) is reported. Unpaired t test was used for statistical analysis. Each dot represents one mouse (WT, n = 6; KO, n = 5). Data represent one out of three independent experiments (mean ± s.d.). ***P < 0.001, ****P < 0.0001, n.s., not significant.

- ExtendedDataFigure9.tif
Extended Data Fig. 9 Transcriptomic changes in ALKBH5 deficient γδ T cell progenitors. a, Numbers of genes that are up-regulated and down-regulated in ALKBH5 deficient γδ T cell progenitors. Differently expressed genes based one P-value (P < 0.05). b, Gene Ontology (GO) enrichment analysis of biological process for up-regulated genes in ALKBH5-deficient γδ T cell precursors.

- ExtendedDataFigure9.tif
Extended Data Fig. 9 Transcriptomic changes in ALKBH5 deficient γδ T cell progenitors. a, Numbers of genes that are up-regulated and down-regulated in ALKBH5 deficient γδ T cell progenitors. Differently expressed genes based one P-value (P < 0.05). b, Gene Ontology (GO) enrichment analysis of biological process for up-regulated genes in ALKBH5-deficient γδ T cell precursors.

- ExtendedDataFigure10.tif
Extended Data Fig. 10 Transcriptomic changes in ALKBH5 deficient mature γδ T cells. a, Numbers of genes that are up-regulated and down-regulated in ALKBH5 deficient mature γδ T cells. Differently expressed genes based one P-value (P < 0.05). b, Heatmap showing Jagged/Notch signaling-related genes expression across different samples. c,d, Gene Ontology (GO) enrichment analysis of biological process for down-regulated (c) and up-regulated genes (d) in ALKBH5-deficient mature γδ T cells. Data represent three independent experiments combined.

- ExtendedDataFigure10.tif
Extended Data Fig. 10 Transcriptomic changes in ALKBH5 deficient mature γδ T cells. a, Numbers of genes that are up-regulated and down-regulated in ALKBH5 deficient mature γδ T cells. Differently expressed genes based one P-value (P < 0.05). b, Heatmap showing Jagged/Notch signaling-related genes expression across different samples. c,d, Gene Ontology (GO) enrichment analysis of biological process for down-regulated (c) and up-regulated genes (d) in ALKBH5-deficient mature γδ T cells. Data represent three independent experiments combined.

- SupplementaryTable1.xlsx
Supplementary Table 1

- SupplementaryTable1.xlsx
Supplementary Table 1

- SupplementaryTable2.xlsx
Supplementary Table 2

- SupplementaryTable2.xlsx
Supplementary Table 2

- SupplementaryTable3.xlsx
Supplementary Table 3

- SupplementaryTable3.xlsx
Supplementary Table 3

- SupplementaryTable4.csv
Supplementary Table 4

- SupplementaryTable4.csv
Supplementary Table 4

- SupplementaryTable5.xlsx
Supplementary Table 5

- SupplementaryTable5.xlsx
Supplementary Table 5

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Posted 01 Dec, 2020

###### Community comments: 1

Posted 01 Dec, 2020

###### Community comments: 1

γδ T cells are abundant T cell population at the mucosa and are important in providing immune surveillance as well as maintaining tissue homeostasis. However, despite γδ T cells origin in thymus, detailed mechanisms regulating γδ T cell development remain poorly understood. *N*6-methyladenosine (m6A) represents one of the most common post-transcriptional modifications of mRNA in mammalian cells, but whether it plays a role in γδ T cell biology is still unclear. Here we show that depletion of m6A demethylase ALKBH5 in lymphocytes specifically induces an expansion of γδ T cells, which confers enhanced protection against gastrointestinal *S. typhimurium* infection. Mechanistically, loss of ALKBH5 favors the development of γδ T cell precursors by increasing the abundance of m6A RNA modification in thymocytes, which further reduces the expression of several target genes including Notch signaling components *Jagged1* and *Notch2*. As a result, impairment of Jagged1/Notch2 signaling contributes to enhanced proliferation and differentiation of γδ T cell precursors, leading to an expanded mature γδ T cell repertoire. Taken together, our results indicate a checkpoint role of ALKBH5 and m6A modification in the regulation of γδ T cell early development.

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