STAT5B leukemic mutations, altering SH2 tyrosine 665, have opposing impacts on immune gene programs

Abstract

STAT5B is a vital transcription factor for lymphocytes. Here, function of two STAT5B mutations from human T cell leukemias: one substituting tyrosine 665 with phenylalanine (STAT5B^Y665F), the other with histidine (STAT5B^Y665H) was interrogated. In silico modeling predicted divergent energetic effects on homodimerization with a range of pathogenicity. In primary T cells in vitro STAT5B^Y665F showed gain-of-function while STAT5B^Y665H demonstrated loss-of-function. Introducing the mutation into the mouse genome illustrated that the gain-of-function Stat5b ^Y665F mutation resulted in accumulation of CD8⁺ effector and memory and CD4⁺ regulatory T-cells, altering CD8⁺/CD4⁺ ratios. In contrast, STAT5B^Y665H 'knock-in' mice showed diminished CD8⁺ effector and memory and CD4⁺ regulatory T cells. In contrast to wild-type STAT5, the STAT5B^Y665F variant displayed greater STAT5 phosphorylation, DNA binding and transcriptional activity following cytokine activation while the STAT5B^Y665H variant resembled a null. The work exemplifies how joining in silico and in vivo studies of single nucleotides deepens our understanding of disease-associated variants, revealing structural determinants of altered function, defining mechanistic roles, and, specifically here, identifying a gain-of function variant that does not directly induce hematopoietic malignancy.

Fig. 1. STAT5B SH2 dimerization modeled by AlphaFold3.

A Schematic of human STAT5B protein domains showing the locations of tyrosine 665 (Y665) and phospho-tyrosine 699 (pY699). B Structure of the human STAT5B SH2 homodimer generated by AlphaFold3. Binding pockets of key residues pY699 (blue) and F711 (red) are indicated. The STAT5B model shows the hydrophobic binding pocket containing key residue Y665 (magenta). C Structure of the human STAT5B SH2 homodimer generated by AlphaFold3 with residues colored red relative to their importance to the binding interface as predicted by COORDinator. D Structure of the human STAT5B SH2 monomer in its dimeric conformation generated by AlphaFold3 with residues colored red relative to their importance to stabilization of the C-terminal tail as predicted by COORDinator. E Model of the human STAT5B SH2 homodimer with tyrosine, histidine, or phenylalanine at position 665, as predicted by AlphaFold3. F The STAT5B model highlights the intramolecular interaction between F711 and the hydrophobic binding pocket containing key residue Y665. G Comparison of the predicted energetic consequences of the F711A, Y665F, and Y665H mutations on stabilization of the C-terminal tail. Relative mutational effects are annotated using ∆∆G, in arbitrary units; stabilizing mutations have a negative value, and destabilizing mutations have a positive value.

Fig. 2. Transcriptomes activated by wild-type and STAT5B mutations in Stat5a/b-null T cells.

A Schematic of the experimental approach to assess the impact of STAT5B^Y665 mutations on naïve CD4 T cells from lymph nodes and spleens of wild-type or STAT5A/B-deficient mice. B The expression level of Stat5b gene transduced with retrovirus encoding STAT5B wild-type Y665, Y665F or Y665H. C Flow cytometry contour plots showing CD4 cell population (n = 3). D Venn diagram displaying the number of significantly induced genes by retroviral vectors encoding Y665F, Y665H and N642H mutations compared to wild type. Cells were stimulated with IL-2 (n = 3). E Heatmaps showing fold changes of significantly upregulated genes between N642H, Y665F and Y665H on Stat5b (Y665) as 1 in STAT5A/B deficient T cells. F Heatmap of genes expressed at significantly higher levels in each sample and significantly enriched in Gene Ontology (GO) terms. G Dot plots of the normalized read counts for mRNA levels of five genes regulated by STAT5B. Results are shown as the means ± SEM of independent biological replicates. P-value are from one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.0001, ****P < 0.0001.

Fig. 3.. Altered T cell distribution in spleens of Stat5b mutant mice.

A Schematic illustration of the experimental approach. The Stat5^Y665 variants were introduced into mouse genome, and T cells and stem cells from immune system organs. Intact or cytokine stimulated cells were subjected to western blot, RNA-seq, scRNA-seq, FACS and ChIP-seq analyses. B Kaplan-Meier curves exhibiting the survival rate. Y665, n = 101; Y665H, n = 187; Y665F^+/−, n = 217; Y665F^−/−, n = 46. C Images of spleens from WT and mutant mice. D Box plots showing total cell number in spleen from wild-type and mutant mice analyzed via flow cytometry (Y665, n = 8; Y665H, n = 5; Y665F, n = 3). P-value are from one-way ANOVA with Tukey’s multiple comparisons test. Median, middle bar inside the box; IQR, 50% of the data; whiskers, 1.5 times the IQR. E Percentages of B cells, CD4 T cells, CD8 T cells, NK cells and neutrophil were calculated using FACS. F-G Percentages of CD8 and CD4 subpopulations. P-value are from two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.0001, ****P < 0.0001. H Count of lymphocytes in peripheral blood from 7–10-week-old adult wild-type and mutant mice. Results are shown as the median of independent biological replicates (Y665, n = 25; Y665H, n = 24; Y665F, n = 16). I and J Numbers of CD4+ and CD8+ T cells identified by flow cytometry. Results are shown as the median of independent biological replicates. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test.

Fig. 4.. Immune dynamics in spleen analyzed using scRNA-seq.

A UMAP projection of lymph node from WT and mutant mice, colored by cell population (n = 1 combined sample from three mice of each group). B Heatmap of cell population. C Frequencies of CD8 subtypes: Tcm (central memory T cells), Tem (effector memory T cells), and NKT (NK-like T cells). D Dot plot of differentially expressed genes related to cytokine signaling and T cell activation in CD8 T cell subsets. E Frequencies of CD4 subtypes: TRM (tissue resident memory T cells), Th (helper T cells), and Treg (regulatory T cells). F-G Dot plot of differentially expressed genes related to homeostasis (F) and inflammatory response (G) in regulatory CD4 T cell subpopulations.

Fig. 5.. Opposing transcriptional activity of STAT5B^Y665F and STAT5B^Y665H at steady-state levels.

A-B Quantification of phospho-STAT5 levels in CD4+ T cells (A) and CD8+ T cells (B) (Y665, n = 8; Y665H, n = 6; Y665F, n = 3). MFI, median fluorescent intensity. P-value are derived from two-way ANOVA with Tukey’s multiple comparisons test. **P < 0.01, ***P < 0.0001, ****P < 0.0001. C Heatmaps depicting fold changes of significantly regulated in Stat5b^Y665F and Stat5b^Y665H mice. D Heatmaps depicting fold changes of significantly upregulated and enriched genes in unstimulated or stimulated T cell from wild-type and Stat5b^Y665F mice (IL-2/7 stimulated Y665, n = 4; IL-2/7 stimulated Y665F, n = 4; IL-2/7 stimulated Y665F, n = 3). E Gene categories expressed at significantly higher levels and depending on STAT5 binding in stimulated T cells from STAT5B^Y665F mice compared to WT mice. F and H Gene categories expressed at significantly higher levels and depending on STAT5 binding in STAT5B^Y665F mice compared to STAT5B^Y665H mice. G and I Heatmaps showing z-score of significantly upregulated and enriched genes in T cell activation (c) and apoptosis (e) between STAT5B^Y665H and STAT5B^Y665F mice (Y665H, n = 5; Y665F, n = 7).

Fig. 6.. Inverse impact of STAT5B^Y665F and STAT5B^Y665H on the establishment of transcription enhancers and immune programs.

A Coverage plots displaying the patterns of STAT5B binding, H3K27ac marks and Pol II loading on promoters of genes with or without STAT5B binding (blue, Y665H; red, Y665F; n = 2–3). B Distribution of STAT5B binding peaks across genomic regions. ChIP-seq signals within ± 3kbp of STAT5B binding regions identified in IL-2/IL-7 stimulated splenic T cells of Y665F mice are shown as pie chart. C Enhancer cluster analysis using IL-2/7 stimulated T cells of Y665F mice. Y-axis indicates the normalized peak score (enhancer cluster score) of STAT5B binding, whereas the x-axis represents the ranking of peaks. No enhancer clusters were identified in Y665H mice. D STAT5B binding, H3K27ac and Pol II loading at gene loci regulated by wild type and mutant STAT5B in IL-2/7 stimulated T cells.