Fig. 5

Lmo4 overexpression enhances STAT3 phosphorylation and STAT3 target gene expression. a qPCR of Tcf7, Zfp36, Socs3, and Junb mRNA in pmel-1 Thy1.1⁺ and Lmo4-Thy1.1⁺ CD8⁺ T cells 5 d after transfer of 10⁵ pmel-1 CD8⁺ T cells transduced with either Thy1.1⁺ or Lmo4-Thy1.1⁺ into wild-type mice infected with gp100-vv. b GSEA showing positive enrichment of genes upregulated in response to IL-21 in CD4⁺ T cells⁴² (left panel) and genes displaying one or more STAT3 binding motifs (GSEA C3:STAT3_02) (right panel) in pmel-1 Lmo4-Thy1.1⁺ CD8⁺ T cells harvested as in (a). c Gene regulatory network that interfaces with Stat3 in pmel-1 Thy1.1⁺ vs. Lmo4-Thy1.1⁺ CD8⁺ T cells. Genes upregulated in Thy1.1 cells are shown in gray color gradient, whereas those overexpressed in Lmo4-Thy1.1 cells are marked in blue gradient. Network was created using Cytoscape software and STRING database. Line thickness of network edges indicates the strength of data support for each interaction based on the information available in the STRING database. d Immunoblot of pSTAT3 and STAT3 (control) in Thy1.1 and Lmo4-Thy1.1 overexpressing T cells at 0, 0.5, and 2 h after adding IL-6 (left panel), IL-10 (middle panel) or IL-21 (right panel) to the cell culture. e q-PCR of Tcf7 (left panel), Zfp36 (middle left panel), Socs3 (middle right panel), and Junb (right panel) mRNA in Lmo4-Thy1.1 relative to Thy1.1 overexpressing T cells after culture with IL-6 or IL-21 for the indicated time. f Immunoblot of Lmo4-Thy1.1 CD8⁺ T cell lysates and anti-JAK1 immunoprecipitates blotted with JAK1 and LMO4 specific antibodies. Anti-IgG2a immunoprecipitates were used as controls. g Flow cytometry histograms showing IL21R expression in pmel-1 CD8⁺ T cells to assess the knockout efficiency compared to controls. h, i Percentages of CD62L⁺KLRG1^- (h) and CD62L^-KLRG1⁺ (i) splenic pmel-1 CD8⁺ T cells 5 d after transfer of either 10⁵ Thy1.1⁺Il21rKO or Lmo4-Thy1.1⁺Il21rKO pmel-1 Ly5.1⁺ CD8⁺ T cells into wild-type mice infected with gp100-vv (n = 5 mice/group). Results are relative to Thy1.1⁺Thy1.2KO and Lmo4-Thy1.1⁺Thy1.2KO control cells, respectively. j Percentages of pmel-1 CD8⁺ T cells in the lymph nodes 5 d after transfer as in (h, i). k Cartoon depicting the potential interaction of LMO4 with the IL-21-STAT3 signaling pathway. LMO4 binds to JAK1, promoting STAT3 phosphorylation and the expression of target genes such as Tcf7, Socs3, Junb, and Zfp36 to boost memory responses (*P < 0.05; ** P < 0.001, h, i, unpaired two-tailed Student’s t-test; j, ANOVA test, b Kolmogorov–Smirnov test). (k) was created with BioRender.com