An increased proportion of progesterone receptor A in peripheral B cells from women who ultimately underwent spontaneous preterm birth.

The enormous challenge in unraveling the etiology of preterm birth (PTB) is to understand the complex interactions between gestational hormones, the immune system and reproductive tissues. PTB can be divided into spontaneous PTB (sPTB) and medically-indicated PTB, e.g. due to preeclampsia (PE) or HELLP syndrome. Progesterone (P4), important for establishment and maintenance of pregnancy, exerts anti-inflammatory effects. The impact of P4 on B cells and its support of maintaining maternal-fetal tolerance is widely unexplored. Therefore, we aimed to determine whether B cells express the progesterone receptor (PR) and to dissect a possible role of PR+ B cells in PTB. We found enhanced IL-6, IL-21 and TNF-α concentrations in maternal plasma in patients with sPTB and PE/HELLP compared to term delivery (TD), accompanied by enhanced PR-A expression by CD19+ B cells. In a second phase of the study, we recruited patients with imminent PTB (iPTB) and controls. Samples were collected at hospital admission and to a later time point, then divided into iPTB patients who delivered preterm and patients whose PTB was prevented. Within the group of iPTB patients, we observed very clear differences: enhanced levels of pro-inflammatory cytokines and increased percentages of PR-A+CD19+ B cells were found in iPTB patients that delivered preterm compared to patients who did not deliver preterm. We conclude that PTB is associated with the activation of an inflammatory pathway leading to the induction of PR-A by B cells. This might further trigger inflammation, result in the break of maternal-fetal tolerance and induce delivery.

Depletion of Foxp3+ regulatory T cells but not the absence of CD19+IL-10+ regulatory B cells hinders tumor growth in a para-orthotopic neuroblastoma mouse model.

Adaptive immune cells with regulatory function reportedly mediate immune escape in a variety of tumors. Little is known regarding the relevance of the most prominent regulatory cell populations, namely Foxp3+ T regulatory cells (Tregs) and CD19+IL-10+ B regulatory cells (Bregs), for neuroblastoma (NB) survival. After establishing a novel immunocompetent syngeneic NB mouse model where orthotopic tumors can be generated after intrarenal injection of NB975A cells, we studied the importance of Tregs and Bregs in Foxp3-DTR mice whose Tregs can be depleted by diphtheria toxin (DT) application as well as in CD19-specific IL-10 deficient mice that lack IL-10+ Bregs (CD19cre+/- × IL-10fl/fl mice). We observed Foxp3 Treg cells in tumors from wild type mice. On the contrary, Bregs or B cells were scarce. Specific depletion of Tregs in Foxp3-DTR mice resulted in an 85% reduction of tumor volume and weight compared to DT-treated wild type mice and untreated Foxp3-DTR mice. In contrast, NB tumor growth was not affected in CD19-specific IL-10 deficient mice. Similarly, mice lacking mature B cells (µMT mice) and CD19 deficient mice (CD19cre mice) showed no change in growth pattern of NB tumors. In Treg-depleted mice, reduced tumor growth was associated with an increased concentration of IFN-gamma, TNF-alpha, IL-4, IL-6, and IL-10 in isolated splenocytes. In summary, transient ablation of Tregs but not absence of Bregs hindered the growth of NB, strongly suggesting the therapeutic potential of targeting Tregs for this aggressive childhood tumor.

Regulatory B Cells Are Decreased and Impaired in Their Function in Peripheral Maternal Blood in Pre-term Birth.

Preterm birth (PTB) is defined as birth before 37 completed weeks of gestation. The causes of PTB are multiple and complex, the underlying pathophysiology being largely unknown. Interferences in the fine-tuned balance of the maternal immune system have been pointed to as one possible cause of PTB. Regulatory B cells (Breg) are part of the adaptive immune response, and recent data suggest that they may contribute to a healthy pregnancy by their regulatory/suppressive function. We investigated the frequency of Breg cells in peripheral blood of women undergoing PTB and control women immediately before giving birth via cesarean section. We detected an enhanced number of B cells, but a reduced number of Breg cells in women delivering preterm. In addition, the percentage of IL-10-producing B cells was decreased in PTB following stimulation with TLR agonists CpG or LPS, alone or combined with CD40L. This was associated with increased levels of pro-inflammatory cytokines in maternal serum. Moreover, isolated maternal B cells before delivering premature babies secreted higher level of the pro-inflammatory cytokine IL-6. No alterations in the frequency of regulatory T cells were found. Our data indicate that alterations in the number and function of Breg cells in peripheral maternal blood contribute to the immunological changes observed in preterm delivery and suggest these cells as important regulators of maternal immune responses.

Chimeric Antigen Receptor-Redirected Regulatory T Cells Suppress Experimental Allergic Airway Inflammation, a Model of Asthma.

Cellular therapy with chimeric antigen receptor (CAR)-redirected cytotoxic T cells has shown impressive efficacy in the treatment of hematologic malignancies. We explored a regulatory T cell (Treg)-based therapy in the treatment of allergic airway inflammation, a model for asthma, which is characterized by an airway hyper-reactivity (AHR) and a chronic, T helper-2 (Th2) cell-dominated immune response to allergen. To restore the immune balance in the lung, we redirected Tregs by a CAR toward lung epithelia in mice upon experimentally induced allergic asthma, closely mimicking the clinical situation. Adoptively transferred CAR Tregs accumulated in the lung and in tracheobronchial lymph nodes, reduced AHR and diminished eosinophilic airway inflammation, indicated by lower cell numbers in the bronchoalveolar lavage fluid and decreased cell infiltrates in the lung. CAR Treg cells furthermore prevented excessive pulmonary mucus production as well as increase in allergen-specific IgE and Th2 cytokine levels in exposed animals. CAR Tregs were more efficient in controlling asthma than non-modified Tregs, indicating the pivotal role of specific Treg cell activation in the affected organ. Data demonstrate that lung targeting CAR Treg cells ameliorate key features of experimental airway inflammation, paving the way for cell therapy of severe allergic asthma.

In neonates S100A8/S100A9 alarmins prevent the expansion of a specific inflammatory monocyte population promoting septic shock.

The high susceptibility of newborn infants to sepsis is ascribed to an immaturity of the neonatal immune system, but the molecular mechanisms remain unclear. Newborn monocytes massively release the alarmins S100A8/S100A9. In adults, these are major regulators of immunosuppressive myeloid-derived suppressor cells (MDSCs). We investigated whether S100A8/S100A9 cause an expansion of monocytic MDSCs (Mo-MDSCs) in neonates, thereby contributing to an immunocompromised state. Mo-MDSCs have been assigned to CD14+/human leukocyte antigen (HLA)-DR-/low/CD33+ monocytes in humans and to CD11b+/Gr-1int/Ly6G-/Ly6Chi cells in mice. We found monocytes with these phenotypes significantly expanded in their respective newborns. Functionally, however, they did not prove immunosuppressive but rather responded inflammatorily to microbial stimulation. Their expansion did not correlate with high S100A8/S100A9 levels in cord blood. Murine studies revealed an excessive expansion of CD11b+/Gr-1int/Ly6G-/Ly6Chi monocytes in S100A9-/- neonates compared to wild-type neonates. This strong baseline expansion was associated with hyperinflammatory responses during endotoxemia and fatal septic courses. Treating S100A9-/- neonates directly after birth with S100A8/S100A9 alarmins prevented excessive expansion of this inflammatory monocyte population and death from septic shock. Our data suggest that a specific population of inflammatory monocytes promotes fatal courses of sepsis in neonates if its expansion is not regulated by S100A8/S100A9 alarmins.-Heinemann, A. S., Pirr, S., Fehlhaber, B., Mellinger, L., Burgmann, J., Busse, M., Ginzel, M., Friesenhagen, J., von Köckritz-Blickwede, M., Ulas, T., von Kaisenberg, C. S., Roth, J., Vogl, T., Viemann, D. In neonates S100A8/S100A9 alarmins prevent the expansion of a specific inflammatory monocyte population promoting septic shock.

Distinct phenotypic features of neonatal murine macrophages.

Neonates rely on their innate immune system. Resident tissue macrophages are considered to be initiators and regulators of the innate immune response and thus, appear to be especially important to neonates. We hypothesized that the phenotype and function of neonatal tissue macrophages differ from their adult counterparts. Peritoneal macrophages from neonatal (<24 h) and adult (6 weeks old) C57BL/6J mice were isolated and analyzed by high-content chipcytometry. After stimulation for 6 h with LPS (0, 1, 10, 100 ng/mL), macrophage transcriptome was analyzed by microarray and cytokine release was measured using multiparametric bead assays. Antigen presenting capacity was compared by T-cell stimulation assays. We observed that neonatal murine peritoneal macrophages are characterized by selective lack of expression of F4/80, MHC class II, and costimulatory molecules (CD80, CD86). Furthermore, we found differences in the transcriptome between neonatal and adult macrophages, unstimulated and after LPS stimulation. Although neonatal macrophages showed a significantly increased secretion of proinflammatory cytokines upon LPS stimulation, their potential to induce T-cell proliferation was significantly reduced. In conclusion, we observed a distinct phenotype of the neonatal macrophage population. The specific functions of this macrophage population could help to understand the excessive inflammatory reactions observed in the very young.

Age-related increase of VGF-expression in T lymphocytes.

VGF is a protein expressed by neurons and processed into several peptides. It plays a role in energy homeostasis and promotes growth and survival. Recently, VGF mRNA was detected in peripheral leukocytes. Since it is known that aging is associated with a decrease in the development and function of neuronal as well as immune cells, we addressed the question whether a peripheral expression of VGF by CD3+ T cells and CD56+ NK cells is correlated with age. Therefore, the frequency of VGF+CD3+ and VGF+CD56+ cells was determined in mentally healthy volunteers aged between 22 and 88. We found an age-dependent increase in the number of VGF+CD3+ T cells that correlated with HbA1c and the body mass index (BMI). VGF-expression by NK cells was age-independent. Blockade of VGF reduced proliferation and secretion of cytokines such as IL-2, IL-17A, IL-1ß, IL-10 and TNF by CD3+ T cells and PBMCs. Rapamycin-mediated T cell blockade significantly reduced the frequency of VGF-expressing T cells. We conclude that VGF contributes to survival and function of peripheral T cells. The age-dependent increase in VGF-expression could serve as mechanism that counterregulates the decrease in functionality of T lymphocytes.