A critical role for STAT3 transcription factor signaling in the development and maintenance of human T cell memory

Abstract

STAT3 transcription factor signaling in specific T helper cell differentiation has been well described, although the broader roles for STAT3 in lymphocyte memory are less clear. Patients with autosomal-dominant hyper-IgE syndrome (AD-HIES) carry dominant-negative STAT3 mutations and are susceptible to a variety of bacterial and fungal infections. We found that AD-HIES patients have a cell-intrinsic defect in the number of central memory CD4(+) and CD8(+) T cells compared to healthy controls. Naive T cells from AD-HIES patients had lower expression of memory-related transcription factors BCL6 and SOCS3, a primary proliferation defect, and they failed to acquire central memory-like surface phenotypes in vitro. AD-HIES patients showed a decreased ability to control varicella zoster virus (VZV) and Epstein-Barr virus (EBV) latency, and T cell memory to both of these viruses was compromised. These data point to a specific role for STAT3 in human central memory T cell formation and in control of certain chronic viruses.

Figure 1. Patients with AD-HIES have fewer central memory T cells

A. The frequencies of naïve (CD27⁺CD45RO⁻), central (CD27⁺CD45RO⁺), and effector (CD27⁻CD45RO^+/−) memory T cells were measured in AD-HIES patients and healthy age-matched controls by flow cytometry. Representative graphs and gating from age-matched subjects. Cells are gated singlet, aqua viability⁻CD3⁺. B. Absolute lymphocyte counts were used to calculate the number of central memory lymphocytes in each population. * p = 0.0083, ** p = 0.0036. C. A concomitant increase in the number of naïve (CD27⁺CD45RO⁻) T cells was observed in AD-HIES patients. Data are compiled from seven independent experiments. *** p = 0.0261, •p = 0.0160. D and E. CD127 expression on central memory (CD27⁺CD45RO⁺) T cells was measured by flow cytometry. D. Representative histograms of CD127 expression on CD27⁺CD45RO⁺ central memory T cells. E. Mean fluorescence intensity of CD127 on the surface of central memory AD-HIES T cells was lower than that of control cells •• p = 0.0317, ••• p = 0.0635. Data are representative of two similar experiments. All error bars represent median values, and significance calculated using a two-tailed Mann-Whitney t test with a 95% confidence interval.

Figure 2. AD-HIES T cells have an intrinsic defect in memory differentiation

A. The frequency of central memory CD4⁺ T cells in a patient mosaic for an AD-HIES STAT3 mutation (G114ASTAT3) and their AD-HIES child. Cells are gated CD3⁺CD4⁺CD27⁺. Naïve (rectangle, CD27⁺CD31⁺CD45RO⁻) and central memory (circle, CD27⁺CD31⁻CD45RO⁺) mosaic AD-HIES T cells were purified by flow cytometry and the frequency of G1145ASTAT3 alleles determined by real-time PCR. B. Chromatographs of STAT3 and a control SNP (intron 13, rs2293152) depict the relative presence of mutant and normal STAT3 alleles in the naïve and memory CD4⁺ population. C. Naïve (CD31⁺CD27⁺CD45RO⁻), central (CD31⁻CD27⁺CD45RO⁺) and effector memory (CD31⁻CD27⁺CD45RO^+/−) CD4⁺ and CD8⁺ T cells were sorted from the mosaic AD-HIES patient and subjected to sequence specific real-time PCR for the G114ASTAT3 allele. Real-time PCR measurements of mutant STAT3 alleles were measured in two independent experiments. D. Naïve (CD19⁺CD27^lo) and memory (CD19⁺CD27^hi) B cells were sorted from the AD-HIES mosaic patient and the frequency of cells with the G1145ASTAT3 allele was measured by real-time PCR. E. Naïve (CD31⁺CD27⁺CD45RO⁺) STAT3-mosaic CD4⁺ T cells were stimulated with irradiated feeder cells, PHA, with or without anti-IL-2Rα (Daclizumab) for ten days and mutant-STAT3 transcripts measured. Data is representative of two independent experiments.

Figure 3. Decrease in central memory T cells in AD-HIES is not due to increased ex vivo apoptosis or decreased in vivo division

A. PBMCs from five AD-HIES and five healthy controls were thawed and cultured in complete RPMI for 48 hours without stimulation or exogenous cytokine. The frequency of AnnexinV⁺7AAD⁻ cells was measured by flow cytometry in naïve (CD27⁺CD45RO⁻), central (CD27⁺CD45RO⁺), and effector (CD27⁻CD45RO^+/−) memory CD4⁺ and CD8⁺ subsets. B to D. Thawed PBMCs were stained for intracellular Ki67 and gated on CD27⁺CD45RO⁺ (central) and CD27⁻CD45RO^+/− (effector) T cells. B. Representative flow cytometry of Ki67 expression within CD4⁺ CD27⁺CD45RO⁺ central memory cells. Graphs depict singlet, aqua viability⁻CD3⁺CD4⁺CD27⁺CD45RO⁺ cells. C and D. Scatter plots depict Ki67 expression within central and effector T cell subsets of AD-HIES and control PBMCs from two independent experiments * p=0.0006, ** p =0.0401, *** p=0.0093. E. Serum levels of IL-2, IL-7, and IL-15 were measured by multiplex cytokine analysis. • p = 0.0014. All error bars represent median values, and significance calculated using a two-tailed Mann-Whitney t test with a 95% confidence interval.

Figure 4. STAT3 mutant naïve T cells have impaired proliferation and in vitro induction of central memory T cell phenotypes

A. Sorted naïve (CD27⁻CD45RO⁻) CD4⁺ T cells were labeled with CFSE and cultured with anti-CD3 and anti-CD28 for five days in the presence of IL-2, IL-7, or IL-2 and IL-7. * p =0.05, ** p = 0.05, *** p = 0.05. B. Sorted naïve (CD27⁻CD45RO⁻) CD8⁺ T cells were labeled with CFSE and cultured with anti-CD3 and anti-CD28 for five days in the presence of IL-2, IL-15, or IL-2 and IL-15. • p = 0.05, •• p = 0.05. All error bars represent median values, and significance was calculated using a one-tailed Mann-Whitney t test with a 95% confidence interval. C and D. After four or five days, blasting or cells were analyzed for CD62L and CD45RO expression to determine differentiation status. Each bar graph represents and individual experiment with a unique control and AD-HIES pair. Different controls and AD-HIES patients were assayed in each experiment.

Figure 5. STAT3 mutant naïve T cells have decreased central memory-related transcription factor expression

A. Naïve (CD27⁺CD45RO⁻) CD4⁺ and CD8⁺ T cells were isolated from AD-HIES patients and controls by flow cytometry and transcript levels measured by real-time PCR. Each point represents the ratio of transcript observed in an AD-HIES patient compared to a corresponding healthy control subject. All data was collected from separate AD-HIES and control patients. B. Naïve (CD27⁺CD45RO⁻), central memory (CD27⁺CD45RO⁺), and effector memory (CD27⁻CD45RO^+/−) CD8⁺ populations were purified by flow cytometry and BCL6 and PRDM1 transcripts were measured by real-time PCR. Each set of CD8⁺ populations was purified from a single individual. Data are representative of four independent experiments with four AD-HIES patients and four control subjects. C. Ratio of BCL6 and PRDM1 transcript levels between AD-HIES and control central and effector memory CD8⁺ T cell populations. Each point represents the ratio of transcript observed in an AD-HIES patient compared to their corresponding normal control.

Figure 6. AD-HIES patients have an increased risk for VZV reactivation and EBV viremia along with defects in virus-specific T cell memory

A. Kaplan-Meier plot of incidence of herpes zoster (VZV reactivation) in AD-HIES cohort. B. Thawed PBMCs were stimulated with inactivated homogenate from VZV infected cells and the sum of IFNγ, TNFα, and IL-2 production by total memory (CD27⁺CD45RO⁺ and CD27⁻CD45RO^+/−) CD4⁺ T cells was measured relative to unstimulated cells. Data are composite of seven independent experiments. * p = 0.0127, ** p = 0.0089. C. The frequency of PBMCs harboring EBV genomes was determined by PCR assay. *** p = 0.0014. D. Thawed PBMCs were stimulated with inactivated lysate from EBV infected cells and IFNγ, TNFα, and IL-2 levels in total memory (CD27⁺CD45RO⁺ and CD27⁻CD45RO^+/−) CD4⁺ and CD8⁺ T cells were measured by flow cytometry. E and F. T cells that produced TNFα and/or IFNγ in response to EBV were analyzed for their surface expression of CD27 and CD45RO. Data compiled from seven independent experiments. • p = 0.004, •• p = 0.0493. All error bars represent median values, and significance calculated using a two-tailed Mann-Whitney t test with a 95% confidence interval.