Signal transducer and activator of transcription 3 (STAT3) mutations underlying autosomal dominant hyper-IgE syndrome impair human CD8(+) T-cell memory formation and function

Abstract

Background: The capacity of CD8(+) T cells to control infections and mediate antitumor immunity requires the development and survival of effector and memory cells. IL-21 has emerged as a potent inducer of CD8(+) T-cell effector function and memory development in mouse models of infectious disease. However, the role of IL-21 and associated signaling pathways in protective CD8(+) T-cell immunity in human subjects is unknown.

Objective: We sought to determine which signaling pathways mediate the effects of IL-21 on human CD8(+) T cells and whether defects in these pathways contribute to disease pathogenesis in patients with primary immunodeficiencies caused by mutations in components of the IL-21 signaling cascade.

Methods: Human primary immunodeficiencies resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Lymphocytes from patients with loss-of-function mutations in signal transducer and activator of transcription 1 (STAT1), STAT3, or IL-21 receptor (IL21R) were used to assess the respective roles of these genes in human CD8(+) T-cell differentiation in vivo and in vitro.

Results: Mutations in STAT3 and IL21R, but not STAT1, led to a decrease in multiple memory CD8(+) T-cell subsets in vivo, indicating that STAT3 signaling, possibly downstream of IL-21R, regulates the memory cell pool. Furthermore, STAT3 was important for inducing the lytic machinery in IL-21-stimulated naive CD8(+) T cells. However, this defect was overcome by T-cell receptor engagement.

Conclusion: The IL-21R/STAT3 pathway is required for many aspects of human CD8(+) T-cell behavior but in some cases can be compensated by other signals. This helps explain the relatively mild susceptibility to viral disease observed in STAT3- and IL-21R-deficient subjects.

Keywords: AD-HIES; Autosomal dominant hyper-IgE syndrome; B-cell lymphoma; BCL; CTV; CellTrace Violet; Central memory T; EOMES; Effector memory T; Effector memory T cells expressing CD45RA; Eomesodermin; IL-21; IL-21 receptor; IL-21R; LCMV; Lymphocytic choriomeningitis virus; PB; PID; Peripheral blood; Primary immunodeficiency; STAT; STAT1; STAT3; Signal transducer and activator of transcription; T(CM); T(EM); T(EMRA); T-cell receptor; TCR; differentiation; human CD8(+) T cells; memory.

Figure 1. IL-21 predominately activates STAT1, STAT3 and STAT5 in human CD8⁺T cells

(A-C) Naïve CD8⁺T cells were activated for 4 days with TAE beads and recultured with cytokines for 30 min to determine phosphorylation of STAT1, 3 and 5. Histograms show nil or cytokine cultures and are representative of 4 experiments. (D-F) Graphs represent fold increase in MFI (mean ± SEM; n=4) of cells stimulated with cytokine over nil cultures. The dashed lines indicate a fold-change of 1 i.e. no change.

Figure 2. Cytokine-induced proliferation is impaired in IL-21R-deficient, but not STAT1- or STAT3-deficient, naïve CD8⁺ T cells

Naïve CD8⁺ T cells were cultures with cytokines only and (A) the numbers of live cells were determined (mean ± SEM, n=5). (B, C) Histograms show representative CTV profiles of cells stimulated with IL-21/IL-15. Graph in (B) shows the number of divided cells in IL-21/IL-15 cultures (mean ± SEM; n=5). (D) Each bar represents an individual patient or normal donor for experiments using cells from STAT1- and IL-21R-deficient patients. * P<0.05; **, P<0.001; *** P<0.001.

Figure 3. Cytokine-induced expression of granzyme B is impaired in IL-21R-deficient or STAT3-deficient naïve CD8⁺ T cells

Naïve CD8⁺ T cells were cultured with cyokines only and (A and B) graphs depict fold increase (mean ± SEM; n=5) in MFI of granzyme B over normal cells cultured with media alone (A). Each bar graph in (B) represents mean and range of duplicate cultures from an individual patient or normal donor for experiments using cells from STAT1- and IL-21R deficient patients. (A) and (C) are representative plots of cells from normal donors or the indicated patients stimulated with IL-21/IL-15 and normal donor cells from unstimulated cultures. ** P<0.001; *** P<0.001.

Figure 4. STAT3-deficient naïve CD8⁺ T cells proliferate normally in response to TCR engagement and activating cytokines

Naive CD8⁺ T cells from healthy donors (n=6-8 [A-C], 3 [D], 5 [E]), STAT3-deficient (A-C; n=6-8), STAT1-deficient (D; n=3); or IL-21R-deficient (E; n=2) patients, were cultured with TAE beads alone or together with cytokines. Total numbers of live cells that had entered division (A, D and E) from each culture were determined (mean ± SEM). (B) Histograms show representative CTV. Numbers give % of divided cells (mean ± SEM). (C-E) Percentage of CD8⁺ T cells in each division was determined. * P<0.05; ** P<0.001; *** P<0.001

Figure 5. IL-21 induced granzyme B production is intact in TCR-stimulated STAT3-deficient CD8⁺ T cells

Naive CD8⁺ T cells from (A) STAT3-deficient (n=6-8), (B) STAT1-deficient (n=3), (C) IL-21R-deficient (n=2) patients or healthy donors (n=6-8), were cultured with TAE beads alone or together with cytokines. Representative histograms of granzyme B expression (filled – normal, coloured lines – patients) are depicted in (A-C). Graphs in (D) show fold-increase (mean ± SEM) in MFI of granzyme B expression by cytokine-stimulated normal and patient cells over those cultured with TAE beads alone.

Figure 6. Mutations in STAT3 but not STAT1 impair generation of effector/memory CD8⁺ T cells in vivo

PB from healthy donors (n=46 or 51), STAT3-deficient (n=13), STAT1-deficient (n=5 or 6) or IL-21R-deficient (n=3) patients were assessed for the percentage of (A) total CD8⁺ T cells or (B) naïve, T_CM, T_EM and TEMRA cells. Each symbol corresponds to an individual donor or patient and lines represent means. Histograms and dot plots are from one representative donor or patient. ** P<0.001; *** P<0.001

Figure 7. CD8⁺ T cells from STAT3-deficient display a more activated phenotype

Subsets of naïve, T_CM, T_EM and T_EMRA CD8⁺ T cells in PB of healthy donors (n=15), STAT3-deficient (n=7), STAT1-deficient (n=3) and IL21R-deficient (n=2) patients were assessed for expression of 2B4, CD57, CD95, CD127, CX3CR1, CD11a, CD11b, granzyme B or perforin. Graphs represent fold-change in MFI of the molecules relative to naïve cells, or the % of positive cells. **, P<0.001; ***, P<0.001