These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

Live attenuated vaccines are generally highly efficacious and often superior to inactivated vaccines, yet the underlying mechanisms of this remain largely unclear. Here we identify recognition of microbial viability as a potent stimulus for follicular helper T cell (T cell) differentiation and vaccine responses. Antigen-presenting cells (APCs) distinguished viable bacteria from dead bacteria through Toll-like receptor 8 (TLR8)-dependent detection of bacterial RNA. In contrast to dead bacteria and other TLR ligands, live bacteria, bacterial RNA and synthetic TLR8 agonists induced a specific cytokine profile in human and porcine APCs, thereby promoting T cell differentiation. In domestic pigs, immunization with a live bacterial vaccine induced robust T cell and antibody responses, but immunization with its heat-killed counterpart did not. Finally, a hypermorphic TLR8 polymorphism was associated with protective immunity elicited by vaccination with bacillus Calmette-Guérin (BCG) in a human cohort. We have thus identified TLR8 as an important driver of T cell differentiation and a promising target for T cell-skewing vaccine adjuvants.

Local and peripheral immune responses are activated after ischemic stroke. In our present study, we investigated the temporal distribution, location, induction, and function of regulatory T cells (Tregs) and the possible involvement of microglia, macrophages, and dendritic cells after middle cerebral artery occlusion (MCAO). C57BL/6J and Foxp3(EGFP) transgenic mice were subjected to 30 minutes MCAO. On days 7, 14, and 30 after MCAO, Tregs and antigen presenting cells were analyzed using fluorescence activated cell sorting multicolor staining and immunohistochemistry. A strong accumulation of Tregs was observed on days 14 and 30 in the ischemic hemisphere accompanied by the elevated presence and activation of microglia. Dendritic cells and macrophages were found on each analyzed day. About 60% of Foxp3(+) Tregs in ischemic hemispheres were positive for the proliferation marker Ki-67 on days 7 and 14 after MCAO. The transfer of naive CD4(+) cells depleted of Foxp3(+) Tregs into RAG1(-/-) mice 1 day before MCAO did not lead to a de novo generation of Tregs 14 days after surgery. After depletion of CD25(+) Tregs, no changes regarding neurologic outcome were detected. The sustained presence of Tregs in the brain after MCAO indicates a long-lasting immunological alteration and involvement of brain cells in immunoregulatory mechanisms.

New treatment strategies for inflammatory bowel disease are needed and parasitic nematode infections or application of helminth components improve clinical and experimental gut inflammation. We genetically modified the probiotic bacterium Escherichia coli Nissle 1917 to secrete the powerful nematode immunomodulator cystatin in the gut. This treatment was tested in a murine colitis model and on post-weaning intestinal inflammation in pigs, an outbred model with a gastrointestinal system similar to humans. Application of the transgenic probiotic significantly decreased intestinal inflammation in murine acute colitis, associated with increased frequencies of Foxp3(+) Tregs, suppressed local interleukin (IL)-6 and IL-17A production, decreased macrophage inflammatory protein-1α/β, monocyte chemoattractant protein -1/3, and regulated upon activation, normal T-cell expressed, and secreted expression and fewer inflammatory macrophages in the colon. High dosages of the transgenic probiotic were well tolerated by post-weaning piglets. Despite being recognized by T cells, secreted cystatin did not lead to changes in cytokine expression or macrophage activation in the colon. However, colon transepithelial resistance and barrier function were significantly improved in pigs receiving the transgenic probotic and post-weaning colon inflammation was reduced. Thus, the anti-inflammatory efficiency of a probiotic can be improved by a nematode-derived immunoregulatory transgene. This treatment regimen should be further investigated as a potential therapeutic option for inflammatory bowel disease.

Although mechanisms leading to brain-specific inflammation and T cell activation have been widely investigated, regulatory mechanisms of local innate immune cells in the brain are only poorly understood. In this study, to our knowledge we show for the first time that MHC class II+CD40dimCD86dimIL-10+ microglia are potent inducers of Ag-specific CD4+Foxp3+ regulatory T cells (Tregs) in vitro. Microglia differentially regulated MHC class II expression, costimulatory molecules, and IL-10 depending on the amount of IFN-γ challenge and Ag dose, promoting either effector T cell or Treg induction. Microglia-induced Tregs were functionally active in vitro by inhibiting Ag-specific proliferation of effector T cells, and in vivo by attenuating experimental autoimmune encephalomyelitis disease course after adoptive transfer. These results indicate that MHC class II+CD40dimCD86dimIL-10+ microglia have regulatory properties potentially influencing local immune responses in the CNS.

Taken together, these data demonstrate that the released proteins of larval T. suis have significant immunomodulatory capacities and efficiently dampen allergic airway hyperreactivity. Thus, the therapeutic potential of defined larval E/S proteins should be exploited for the treatment of human allergic disorders.

No abstract

Porcine cytomegalovirus (PCMV), that is actually a porcine roseolovirus (PRV), is a common herpesvirus in domestic pigs and wild boars. In xenotransplantation, PCMV/PRV has been shown to significantly reduce the survival time of pig kidneys and hearts in preclinical trials with different non-human primates. Furthermore, PCMV/PRV has been transmitted in the first pig to human heart xenotransplantation and contributed to the death of the patient. Although transmitted to the recipient, there is no evidence that PCMV/PRV can infect primate cells including human cells. PCMV/PRV is closely related to the human herpesviruses 6 and 7, and only distantly related to the human CMV (HCMV). Antiviral drugs used for the treatment of HCMV are less effective against PCMV/PRV. However, there are well described strategies to eliminate the virus from pig facilities. In order to detect the virus and to eliminate it, highly sensitive detection methods and the knowledge of how, where and when to screen the donor pigs is required. Here, a comparative testing of organs from pigs of different ages using polymerase chain reaction (PCR)-based and immunological methods was performed. Testing young piglets, PCMV/PRV was detected effectively by PCR in blood, bronchoalveolar lavage fluid, tonsils and heart. In adult animals, detection by PCR was not successful in most cases, because the virus load was below the detection limit or the virus was in its latent stage. Therefore, detection of antibodies against selected recombinant proteins corresponding to epitopes detected by nearly all infected animals in a Western blot assay is advantageous. By contrast, immunological testing is not beneficial in young animals as piglets might have PCMV/PRV-specific antibodies obtained from their infected mother via the colostrum. Using a thoughtful combination of PCR-based and immunological methods, detection of PCMV/PRV in donor pigs for xenotransplantation is feasible and a controlled elimination of the virus by early weaning or other methods is possible.