Memory CD4+ T cells lacking expression of CCR7 promote pro-inflammatory cytokine production in patients with diffuse cutaneous systemic sclerosis.

Systemic sclerosis (SSc) is a predominantly T-cell-mediated autoimmune disorder with a characteristic sequence of Th1 and Th2 inflammation resulting in fibrosis. The contribution of differentiated memory T-cell subpopulations and methylation of CpG regions of Th1- or Th2-specific transcription factor genes on the inflammatory cytokine signature in SSc is not well understood. The study aimed to investigate phenotypic differentiation, the cytokine signature, sensitivity of memory T cells to in vitro suppression by autologous regulatory T cells (Tregs), and methylation of Th1- and Th2-specific transcription factor genes in patients with limited (lcSSc) and diffuse cutaneous SSc (dcSSc) compared to healthy donors (HD). Phenotype/intracellular cytokine production and methylation of Th1- and Th2-specific transcription factor genes were determined by flow cytometry and epigenetic analysis, respectively, and compared between patients with lcSSc, dcSSc and HD. Discrimination of CD4+ T cells that lack CCR7 expression revealed that CCR7- CD4+ memory T cells and effectors are producers of intracellular TNFα, IL-13 and IL-4, particularly in dcSSc. A proportional increase in CCR7- memory T cells was demonstrated by SSc-derived CD4+ T-cells after insufficient suppression by Tregs. A higher level of methylation of GATA3 or STAT4 (Th2- and Th1-specific transcription factor genes, respectively) was observed in dcSSc. An abundance of specific CD4+ memory T-cell subpopulations strongly contributes to the production of pro-inflammatory cytokines in dcSSc. Our results suggest that therapeutic concepts should focus more intensively on the memory phenotype to control T cell-mediated inflammation in SSc patients.

Impact of USP8 Gene Mutations on Protein Deregulation in Cushing Disease.

CONTEXT: Cushing disease (CD) is a rare disorder with severe sequels and incompletely understood pathogenesis. The underlying corticotroph adenomas harbor frequently somatic mutations in the ubiquitin-specific peptidase 8 (USP8) gene. These mutations render USP8 hyperactive and prevent client proteins from degradation. OBJECTIVE: To investigate the impact of USP8 mutations on proteins deregulated in CD. DESIGN: One hundred eight pituitary adenomas (75 corticotroph [58 USP8 wild type (WT) and 17 USP8 mutated], 14 somatotroph, and 19 nonfunctioning) were investigated by immunohistochemistry. All evaluated proteins [USP8, arginine vasopressin receptor 1b and 2, corticotropin-releasing hormone receptor, cAMP response element-binding protein (CREB), p27/kip1, cyclin E, heat shock protein 90 (HSP90), orphan nuclear receptor 4, epidermal growth factor receptor, histone deacetylase 2, glucocorticoid receptor, cyclin-dependent kinase 5 and Abelson murine leukemia viral oncogene homolog 1 enzyme substrate 1] were known to be deregulated in CD. Furthermore, AtT20 cells were transfected with USP8 to investigate the expression of possible downstream proteins by immunoblot. RESULTS: Whereas most of the investigated proteins were not differentially expressed, the cell-cycle inhibitor p27 was significantly reduced in USP8 mutated corticotroph adenoma (H-score 2.0 ± 1.0 vs 1.1 ± 1.1 in WT adenomas; P = 0.004). In contrast, the chaperone HSP90 was expressed higher (0.5 ± 0.4 vs 0.2 ± 0.4; P = 0.29), and the phosphorylation of the transcription factor CREB was increased in USP8 mutated adenomas (1.30.5 ± 0.40.9 vs 0.70.5 ± 0.40.7; P = 0.014). Accordingly, AtT20 cells transfected with the USP8 P720R mutant had higher phosphorylated CREB (pCREB) levels than WT transfected cells (1.3 ± 0.14 vs 1 ± 0.23; P = 0.13). CONCLUSIONS: We could demonstrate that USP8 mutations are associated with deregulation of p27/kip1, HSP90, and pCREB. These findings suggest that these proteins are direct or indirect clients of USP8 and could therefore be potential targets for therapeutic approaches in patients with CD.

Significant IFNγ responses of CD8+ T cells in CMV-seropositive individuals with autoimmune arthritis.

BACKGROUND: Latent Cytomegalovirus (CMV) infection accelerates immunosenescence in elderly with reactivations reported in Rheumatoid Arthritis (RA) and abnormal responses towards CMV in Juvenile Idiopathic Arthritis (JIA). OBJECTIVES: Considering the signs of premature T-cell immunosenescence in arthritis patients, the known effect of CMV latency on speeding up many of these signs in an age-dependent manner and the role of CMV on IFNγ-mediated inflammation in healthy elderly and RA, we hypothesized that latent CMV infection accelerates TCR repertoire restriction, loss of CD28, peripheral T-cell proliferation and aberrant IFNγ responses in arthritis patients. STUDY DESIGN: Unspecific and CMVpp65-specific IFNγ responses were investigated in peripheral CD8+ T-cells in RA or JIA patients and healthy, age-matched controls. RESULTS: Despite higher prevalence and concentrations of IgG-anti-CMV, arthritis patients showed lower unspecific IFNγ production, lower CD69-mediated activation and lower CD8+ T-cell proliferation. CMV-seropositive RA patients showed higher intracellular IFNγ production and increased proportions of CD28-CD8+ T-cells after specific CMVpp65 long-term stimulation which was not altered by in vitro blockade of TNFα or IL-6. A skewed TCR repertoire towards oligoclonality and less polyclonality was found in JIA. DISCUSSION: CMVpp65-specific IFNγ production with expansion of CD28-CD8+ T-cells suggests an efficient control of latent CMV regardless of immunosuppressive therapy or in vitro blockade of TNFα or IL-6 in CMV-seropositive arthritis patients. Increased IgG-anti-CMV antibody concentrations and increased proportions of intracellular IFNγ-producing CMVpp65-specific CD8+ T-cells in long-term cultures propose a possibly role of endogenous CMV reactivations boosting antibody levels and a higher possibly CMV-driven IFNγ-mediated inflammatory potential of CD8+ T-cells in arthritis patients.

Expression of 11beta-hydroxysteroid-dehydrogenase type 2 in human thymus.

11beta-hydroxysteroid-dehydrogenase type 2 (11ß-HSD2) is a high affinity dehydrogenase which rapidly inactivates physiologically-active glucocorticoids to protect key tissues. 11ß-HSD2 expression has been described in peripheral cells of the innate and the adaptive immune system as well as in murine thymus. In absence of knowledge of 11ß-HSD2 expression in human thymus, the study aimed to localize 11ß-HSD2 in human thymic tissue. Thymic tissue was taken of six healthy, non-immunologically impaired male infants below 12months of age with congenital heart defects who had to undergo correction surgery. 11ß-HSD2 protein expression was analyzed by immunohistochemistry and Western blot. Kidney tissue, peripheral blood mononuclear cells (PBMCs) and human umbilical vein endothelial cells (HUVEC) were taken as positive controls. Significant expression of 11ß-HSD2 protein was found at single cell level in thymus parenchyma, at perivascular sites of capillaries and small vessels penetrating the thymus lobuli and within Hassall's bodies. The present study demonstrates that 11ß-HSD2 is expressed in human thymus with predominant perivascular expression and also within Hassall's bodies. To our knowledge, this is the first report confirming 11ß-HSD2 expression at the protein level in human thymic tissue underlining a potential role of this enzyme in regulating glucocorticoid function at the thymic level.

Reduced varicella-zoster-virus (VZV)-specific lymphocytes and IgG antibody avidity in solid organ transplant recipients.

BACKGROUND: Varicella-zoster-virus (VZV) infection may cause significant morbidity and mortality in immunocompromised patients. So far, only IgG-anti-VZV antibody concentrations were used to estimate immunity against VZV, but the antibody binding strength (avidity) together with VZV-specific cellular responses have not been evaluated in solid organ transplant (SOT) recipients. METHODS: Thus, we assessed the humoral and cellular immune responses to two doses of the VZV vaccine (vacc) and wild-type VZV infection (wt) in 23 kidney (KTx) and 19 liver transplant (LTx) recipients including children and adults compared to 48 healthy controls (HC) for measurement of IgG-anti-VZV relative avidity index (RAI) and frequency of VZV-specific peripheral blood mononuclear cells (PBMCs) in vaccinated individuals using an adapted ELISA and IFN-gamma ELISPOT, respectively. RESULTS: KTx(wt) (median RAI 72.3%) or LTx(wt) (79.2%) and KTx(vacc) (91.0%) or LTx(vacc) (72.5%) showed lower avidities compared to HC(wt) (84.5%) and HC(vacc) (94.0%), respectively, despite equally distributed IgG-anti-VZV concentrations. RAI>60% (high avidity) was detected in all HC, but only in 69.0% of SOT patients. KTx(vacc) (median 64 spot forming units SFU/500,000 PBMCs) and LTx(vacc) (67 SFU) had significantly lower VZV-specific cellular responses compared to HC(vacc) (268 SFU). CONCLUSIONS: The diminished cellular reactivity to VZV has to be considered in SOT patients receiving immunosuppressive treatments when evaluating immunity against VZV. IgG antibody avidity and VZV-specific cellular responses may serve as additional markers to evaluate immunity against VZV in SOT recipients. The role of wild-type exposures and endogenous VZV re-activation on long-term immunity in SOT patients has to be awaited to establish recommendations for vaccine spacing in these patients, considering immunogenicity and safety aspects.