Nadine Unterwalder

Terminally Differentiated CD8+ T Cells Negatively Affect Bone Regeneration in Humans

A subset of T cells inhibits bone regeneration in humans. No Bones About It Sticks and stones may break your bones, but immune cells will not hurt you, at least if Reinke et al. have anything to say about it. The immune system seems to have a hand in everything these days, and bone repair is no exception. T cells have been implicated in modulating bone fracture repair, even in the absence of infection. Reinke et al. take these studies into patients and find that delayed fracture healing correlated with a subset of T cells—terminally differentiated effector memory CD8+ T (TEMRA) cells. The authors examined the number of CD8+ TEMRA cells over time and found that the difference in CD8+ TEMRA cell number in patients with delayed healing reflected the individual’s immune profile, or lifelong response to infection, rather than a more acute, fracture-related event. They specifically found these cells in fracture hematoma, one of the earliest stages of fracture healing. They then took these studies into mice and found that the absence of CD8+ T cells improved bone regeneration, whereas adding CD8+ T cells impaired fracture healing. This mechanistic link supported their association in patients and suggests that these CD8+ TEMRA cells may be targeted or serve as markers for intervention in patients with delayed bone fracture healing. There is growing evidence that adaptive immunity contributes to endogenous regeneration processes: For example, endogenous bone fracture repair is modulated by T cells even in the absence of infection. Because delayed or incomplete fracture healing is associated with poor long-term outcomes and high socioeconomic costs, we investigated the relationship between an individual’s immune reactivity and healing outcome. Our study revealed that delayed fracture healing significantly correlated with enhanced levels of terminally differentiated CD8+ effector memory T (TEMRA) cells (CD3+CD8+CD11a++CD28−CD57+ T cells) in peripheral blood. This difference was long lasting, reflecting rather the individual’s immune profile in response to lifelong antigen exposure than a post-fracture reaction. Moreover, CD8+ TEMRA cells were enriched in fracture hematoma; these cells were the major producers of interferon-γ/tumor necrosis factor–α, which inhibit osteogenic differentiation and survival of human mesenchymal stromal cells. Accordingly, depletion of CD8+ T cells in a mouse osteotomy model resulted in enhanced endogenous fracture regeneration, whereas a transfer of CD8+ T cells impaired the healing process. Our data demonstrate the high impact of the individual adaptive immune profile on endogenous bone regeneration. Quantification of CD8+ TEMRA cells represents a potential marker for the prognosis of the healing outcome and opens new opportunities for early and targeted intervention strategies.

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.

Analyses of phenotypic and functional characteristics of CX3CR1-expressing natural killer cells.

Summary We previously demonstrated a correlation between the frequency of CX3CR1‐expressing human natural killer (NK) cells and disease activity in multiple sclerosis and showed that CX3CR1high NK cells were more cytotoxic than their CX3CR1neg/low counterparts. Here we aimed to determine whether human NK cell fractions defined by CX3CR1 represent distinct subtypes. Phenotypic and functional NK cell analyses revealed that, distinct from CX3CR1high, CX3CR1neg/low NK cells expressed high amounts of type 2 cytokines, proliferated robustly in response to interleukin‐2 and promoted a strong up‐regulation of the key co‐stimulatory molecule CD40 on monocytes. Co‐expression analyses of CX3CR1 and CD56 demonstrated the existence of different NK cell fractions based on the surface expression of these two surface markers, the CX3CR1neg CD56bright, CX3CR1neg CD56dim and CX3CR1high CD56dim fractions. Additional investigations on the expression of NK cell receptors (KIR, NKG2A, NKp30 and NKp46) and the maturation markers CD27, CD62L and CD57 indicated that CX3CR1 expression of CD56dim discriminated between an intermediary CX3CR1neg CD56dim and fully mature CX3CR1high CD56dim NK cell fractions. Hence, CX3CR1 emerges as an additional differentiation marker that may link NK cell maturation with the ability to migrate to different organs including the central nervous system.

Efficient tetanus toxoid immunization on vitamin D supplementation

Background/Objectives:Vitamin D mediates immunomodulatory functions, and its deficiency has been associated with an increased prevalence of immunological diseases. The supplementation of vitamin D might be therapeutically beneficial, for example, in lupus erythematosus patients. However, its affect on established recall immune responses is undefined.Subjects/Methods:In all, 32 individuals were randomized in a placebo controlled, double-blind setting, and received vitamin D (daily 2000 IU) for 10 weeks followed by tetanus toxoid (TT) booster immunization.Results:During vitamin D supplementation the median 25-hydroxyvitamin D serum concentration increased to 80.3 nM, which as expected decreased in the placebo group to 29.1 nM during the ultraviolet-deprived winter months. The TT-specific immunoglobulin G (IgG) boost efficiency was marginal higher in the vitamin D group (P=0.04). The increase of the 25-hydroxyvitamin D levels correlated with the increase of TT–IgG serum concentrations. The induction of specific serum IgA and specific antibody secreting cells was comparable between both groups. Accordingly, the TT-specific and polyclonally triggered T-cell cytokine profiles were stable as well.Conclusions:Vitamin D supplementation was successful and booster immunization induced efficiently specific antibodies titers.

Effects of sotrastaurin, mycophenolic acid and everolimus on human B-lymphocyte function and activation

Humoral rejection processes may lead to allograft injury and subsequent dysfunction. Today, only one B‐cell‐specific agent is in clinical use and the effects of standard and new immunosuppressant substances on B‐cell activation and function are not fully clarified. The impact of sotrastaurin, mycophenolic acid and everolimus on human B‐lymphocyte function was assessed by analysing proliferation, apoptosis, CD80/CD86 expression and immunoglobulin and IL‐10 production in primary stimulated B cells. In addition, B‐cell co‐cultures with pre‐activated T cells were performed to evaluate the effect of the different immunosuppressive agents on T‐cell‐dependent immunoglobulin production. Sotrastaurin did not inhibit B‐cell proliferation, CD80/CD86 expression, and IgG production and had only minor effects on IgM levels at the highest concentration administered. In contrast, mycophenolic acid and everolimus had strong effects on all B‐cell functions in a dose‐dependent manner. All immunosuppressive agents caused decreased immunoglobulin levels in T‐cell‐dependent B‐cell cultures. The data provided here suggest that mycophenolic acid and everolimus, but not sotrastaurin, are potent inhibitors of human B‐lymphocyte function and activation.

Association of TLR3-hyporesponsiveness and functional TLR3 L412F polymorphism with recurrent herpes labialis

HSV-1 persistently infects almost 90% of our population; however, only 30% of the infected subjects suffer from recurrent herpes lesions, most frequently herpes labialis (HL). We hypothesized that variations in toll-like receptor (TLR) functions might contribute to HL susceptibility. In our study, the TLR-2/1,-3, and -7/8 responses of immune cell subsets derived from asymptomatic HSV-1 carriers were compared with responses of subjects with HL history. Remarkably, natural killer (NK) cells isolated from HL subjects showed significantly lower IFN-γ responses selectively to the TLR3 agonist poly(I:C). Furthermore, the TLR3 L412F genetic polymorphism was found to reduce NK cell TLR3-responsiveness and is associated with susceptibility to recurrent HL. The TLR3 response detected in HL total peripheral blood mononuclear cells (PBMCs), however, was not impaired, indicating restoration of NK cell TLR3-deficiency through co-stimulatory functions. In conclusion, our results suggest that decreased TLR3 response of NK cells is associated with HL susceptibility; and potentially explain why symptomatic outbreak of HL usually occurs after stress or prolonged UV light exposure, when host co-stimulatory functions are disturbed.

Identification of T Cell-Mediated Vascular Rejection After Kidney Transplantation by the Combined Measurement of 5 Specific MicroRNAs in Blood.

Background MicroRNAs (miRNAs, miR) hold important roles in the posttranscriptional regulation of gene expression. Their function has been correlated with kidney disease, and they might represent a new class of biomarkers for frequent evaluation of renal graft status. We analyzed their potential in identifying severe T cell–mediated vascular rejection (TCMVR) (Banff 4-II/III) in kidney transplanted patients. Methods Microarray experiments and semiquantitative real-time reverse transcription polymerase chain reaction were performed with total RNA isolated from blood cells of kidney graft recipients. Initial microarray analysis revealed 23 differentially expressed miRNAs distinguishing patients with TCMVR from patients with stable grafts. From these, we validated and further determined the expression of 6 differentially expressed miRNAs and 2 control miRNAs in 161 samples from patients with T cell–mediated rejection (Banff 3-Borderline, Banff 4-I/II/III), Banff-2 antibody-mediated rejection, Banff-5 interstitial fibrosis/tubular atrophy, in samples from stable patients and in samples from patients with urinary tract infection using real-time reverse transcription polymerase chain reaction. Results Expression levels of all 6 candidate miRNAs were significantly downregulated in blood of TCMVR patients compared to the other groups and displayed high sensitivities and specificities for diagnosing TCMVR. The combination of 5 miRNAs, identified by an unbiased multivariate logistic regression followed by cross-validation, enhanced the sensitivity and specificity for the diagnosis of TCMVR after renal transplantation. Conclusions The combined measurement of miRNA-15B, miRNA-16, miRNA-103A, miRNA-106A, and miRNA-107 may help to better identify TCMVR after renal transplantation in a precise and clinically applicable way.

Clinical manifestation of mannose-binding lectin deficiency in adults independent of concomitant immunodeficiency.

Mannose-binding lectin (MBL) mediates important functions within the innate immune system, and its deficiency was associated with infectious complications. However, in adults without concomitant immunodeficiency the clinical relevance of MBL deficiency remains controversial. We analyzed the distribution of MBL deficiency and its association with concomitant immunodeficiency in 228 adult Caucasian patients with a history of recurrent and/or severe infections. Two hundred forty-one unrelated Caucasians without recurrent or severe infections served as control subjects. The frequency of severe MBL deficiency (plasma levels <or= 50 ng/ml) was significantly higher in patients with a history of recurrent and/or severe infections (p < 0.05, odds ratio 2.1, 95% confidence interval 1.1-4.1), and this association was independent of concomitant antibody or cellular immunodeficiency. Our data challenge the view that MBL deficiency in adulthood becomes relevant only in individuals who are immunocompromised for other reasons.

Diminished HLA-DR expression on monocyte and dendritic cell subsets indicating impairment of cellular immunity in pre-term neonates: a prospective observational analysis

Abstract Aims: The risk of neonates for severe infection/sepsis is reciprocally proportional to gestational age and birth weight. As monocytes and dendritic cells (DC) are recognised key antigen-presenting immune cells, we aimed to elucidate whether neonatal age is associated with reduced expression of human-leukocyte antigen-DR (HLA-DR) antigens on subsets of monocytes and DCs. Methods: Forty-three consecutive neonates (20 male, mean gestational age 236.0±26.8 days; mean 1-min Apgar score 7.5±2.0) were included in a monocentric prospective observational analysis. Patients were grouped according to gestational age (n=15 full-term, n=28 pre-term defined as <33 weeks). Ten healthy adult volunteers were assessed also. Flow-cytometric assessment of HLA-DR expression was performed in subsets of peripheral blood myeloid and plasmacytoid DCs (MDC and PDC) and monocytes (CD14brightCD16negative/CD14positiveCD16positive/CD14dimCD16positive). Clinical and routine laboratory data were followed up. Results: At birth, leukocyte counts were increased in full-term neonates. Monocyte counts were significantly increased in neonates when compared with adults (all P<0.05). A significant numerical increase of CD14brightCD16negative and CD14positiveCD16positive monocytes was noted in pre-term and full-term neonates (all P<0.05), while HLA-DR expression in these subsets was significantly diminished (most pronounced in pre-term infants, P<0.0001). MDC and PDC HLA-DR expression was reduced also (all P<0.05). Clinical indices (e.g., pH, days on antibiotics/mechanical ventilation, fever/sepsis) were not found to correlate with immunological indices. Conclusions: We observed a markedly diminished HLA-DR expression on monocyte and DC subsets in pre-term and full-term neonates, which may contribute to impaired antimicrobial defence mechanisms in the early days of life.

Postoperative immunosuppression after open and laparoscopic liver resection: assessment of cellular immune function and monocytic HLA-DR expression.

In an experimental setting, the authors demonstrated a trend toward improved preservation of the immune system after laparoscopic hepatic resection compared with open surgery.