Deficient EBV-specific B- and T-cell response in patients with chronic fatigue syndrome.

Epstein-Barr virus (EBV) has long been discussed as a possible cause or trigger of Chronic Fatigue Syndrome (CFS). In a subset of patients the disease starts with infectious mononucleosis and both enhanced and diminished EBV-specific antibody titers have been reported. In this study, we comprehensively analyzed the EBV-specific memory B- and T-cell response in patients with CFS. While we observed no difference in viral capsid antigen (VCA)-IgG antibodies, EBV nuclear antigen (EBNA)-IgG titers were low or absent in 10% of CFS patients. Remarkably, when analyzing the EBV-specific memory B-cell reservoir in vitro a diminished or absent number of EBNA-1- and VCA-antibody secreting cells was found in up to 76% of patients. Moreover, the ex vivo EBV-induced secretion of TNF-α and IFN-γ was significantly lower in patients. Multicolor flow cytometry revealed that the frequencies of EBNA-1-specific triple TNF-α/IFN-γ/IL-2 producing CD4(+) and CD8(+) T-cell subsets were significantly diminished whereas no difference could be detected for HCMV-specific T-cell responses. When comparing EBV load in blood immune cells, we found more frequently EBER-DNA but not BZLF-1 RNA in CFS patients compared to healthy controls suggesting more frequent latent replication. Taken together, our findings give evidence for a deficient EBV-specific B- and T-cell memory response in CFS patients and suggest an impaired ability to control early steps of EBV reactivation. In addition the diminished EBV response might be suitable to develop diagnostic marker in CFS

EBNA antibody response is reduced in CFS patients.

<p>(A) Serum IgG titers were assessed for healthy controls and CFS patients by ELISA for EBV VCA-IgG (control n = 57, CFS n = 63), (B) EBNA-IgG (control n = 57, CFS n = 63), (C) EBV VCA-IgM (control n = 57, CFS n = 63), (D) CMV-IgG (control n = 32, CFS n = 41) and (E) CMV-IgM (control n = 32, CFS n = 41). Statistical analysis was performed using the two-tailed Mann-Whitney-U test and for EBNA-IgG and EBV VCA-IgM Fisher's exact one-tailed test for association analysis with * p<0.05.</p

EBV-specific antibody secreting cells are reduced in CFS patients.

<p>(A–D) Frequencies of ASCs in healthy controls and CFS patients 7 days after polyclonal stimulation of total PBMCs. Secreted total and specific IgG was assessed with the ELISpot assay. IgG-secreting B cells are shown as frequencies from 1×10⁶ seeded cells for (A) total IgG (control n = 12, CFS n = 17), (B) EBV-lysate-specific IgG (control n = 12, CFS n = 17), VCA-specific IgG (control n = 12, CFS n = 17), IgG against EBNA-1 peptides (control n = 12, CFS n = 16) and (C) HSV- (control n = 8, CFS n = 11) and (D) CMV-lysate-specific IgG (control n = 6, CFS n = 6). (E) Comparison of frequencies of ASCs in polyclonal stimulation of total PBMCs (T-cell dependent) and stimulation of isolated B cells with CD40L (T-cell independent) in CFS patients for total IgG, EBV-lysate, (n = 8), VCA or EBNA-1 peptides (n = 6). Statistical analysis was performed using the two-tailed Mann-Whitney-U test with * p<0.05.</p

Latent EBV can be detected more frequently in CFS patients.

<p>(A) EBV DNA was analyzed via nested real-time PCR in total PBMCs of 20 healthy donors and CFS patients for EBER-1. EBER-copies were calculated in accordance to Namalwa standard. (B) BZLF-1 RNA was analyzed via nested real-time PCR in total PBMCs of 20 healthy donors and CFS patients but no BZLF-1 cDNA was detected. cDNA of EBV cell line 293T/B95-8 was used as positive control. Statistical analysis was performed using the one-tailed Mann-Whitney-U test with ** p<0.01.</p

CFS patients show reduced EBV-specific memory T-cell response.

<p>(A) Comparison of cytokine producing CD4⁺ (upper panels) and CD8⁺ T cells (lower panels) of CFS patients and healthy controls after 10 days of stimulation with EBNA-1 (left panel, Control n = 17, CFS n = 23). Boolean gating strategy was applied to analyze IFN-γ/TNF-α/IL-2 triple, IFN-γ/TNF-α double, and IFN-γ and TNF-α single cytokine producing T cells after intracellular staining of isolated PBMCs incubated with Brefeldin A for 16 h. Stimulation with CMV pp65 (right panel, Control n = 7, CFS n = 5) is shown for IFN-γ/TNF-α/IL-2 triple, and IFN-γ single cytokine producing T cells. (B) Frequencies of PD-1 expression were analyzed for IFN-γ/TNF-α double producing CD4⁺ and CD8⁺ T cells after 10 days of stimulation with EBNA-1 or pp65 (n = 8). Statistical analysis was performed using the two-tailed Mann-Whitney-U test with ** p<0.01.</p

CFS patients show diminished cytokine response against EBV.

<p>Whole blood of healthy controls and CFS patients was analyzed by Multiplex-Immunoassay for (A) IFN-γ production after stimulation with either EBV-lysate (control n = 29, CFS n = 22), EBNA-1 peptide (control n = 24, CFS n = 11) or SEB (control n = 21, CFS n = 11) and (B) after EBV-lysate stimulation for TNF-α (control n = 29, CFS n = 22), IL-2 (control n = 29, CFS n = 22) and IL-10 (control n = 25, CFS n = 13). Statistical analysis was performed using the two-tailed Mann-Whitney-U test with * p<0.05 and *** p<0.001.</p

EBNA-1-IgG is reduced in a subset of patients but total IgG and B-cell subpopulations are not different in EBNA-1-IgG positive and -negative CFS patients.

<p>(A) Serum IgG titers were assessed in CFS patients for EBV-IgG and EBV-EBNA-1-IgG (n = 387), (B–E) EBNA-1 negative (neg, n = 7) and positive (pos, n = 8) CFS patients were compared for (B) total IgG, (C) the absolute numbers of CD19⁺ B cells/nl blood, (D) frequencies of IgD⁺IgM⁺CD27⁺ marginal zone B cells, and (E) frequencies of IgD⁻CD27⁺ class switched memory B cells. Statistical analysis was performed using the two-tailed Mann-Whitney-U test and for IgG Fisher's exact one-tailed test for association analysis with *** p<0.0001.</p

Cellular Players and Role of Selectin Ligands in Leukocyte Recruitment in a T-Cell-Initiated Delayed-Type Hypersensitivity Reaction

Delayed-type hypersensitivity (DTH) reactions are characterized by a strong cellular infiltrate, including neutrophils, macrophages, and T lymphocytes. In all these cell types, both E- and P-selectin-dependent adhesion pathways play a significant role in recruitment into the inflamed skin. Accordingly, inhibition of selectin-mediated interactions (eg, by antibodies) results in impairment of acute DTH reactions. However, whether inhibition of a specific cell type is responsible for the anti-inflammatory effect or whether all leukocytes are affected remains unclear. To address this question, we used fucosyltransferase-VII knockout mice that lack functional selectin ligands as either donors or recipients in a DTH model elicited by Th1 cell and antigen transfer. We found that selectin-mediated adhesion is required by Th1 effector cells to enter the DTH reaction site and, additionally, to elicit the DTH reaction. On the other hand, elimination of selectin binding in the recipient’s neutrophils and macrophages by use of fucosyltransferase-deficient mice receiving wild-type Th1 effector cells resulted in a strongly reduced infiltration of neutrophils and macrophages but unimpaired footpad swelling. These findings demonstrate a major role for both E- and P-selectin in the recruitment of different leukocyte cell types. However, only the presence of selectin ligands on T cells was critical for the inflammatory reaction. These findings reveal T cells as the predominant targets for selectin blockade that aim to suppress skin inflammation

Autoregulation of Th1-mediated inflammation by twist1

The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor {kappa}B (NF-{kappa}B)–dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We show that twist1 is expressed by activated T helper (Th) 1 effector memory (EM) cells. Induction of twist1 in Th cells depended on NF-{kappa}B, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 was transient after T cell receptor engagement, and increased upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression was characteristic of repeatedly restimulated EM Th cells, and thus of the pathogenic memory Th cells characteristic of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohn's disease or ulcerative colitis expressed high levels of twist1. Expression of twist1 in Th1 lymphocytes limited the expression of the cytokines interferon-{gamma}, IL-2, and tumor necrosis factor-{alpha}, and ameliorated Th1-mediated immunopathology in delayed-type hypersensitivity and antigen-induced arthritis