SummaryBackgroundRecently, it has been established that pollen grains contain Th2‐enhancing activities besides allergens.ObjectiveThe aim of this study was to analyse whether pollen carry additional adjuvant factors like microbes and what immunological effects they may exert.MethodsTimothy pollen grains were collected and disseminated on agar plates, and the growing microorganisms were cultivated and defined. Furthermore, the immunologic effects of microbial products on DC and T cell responses were analysed.ResultsA complex mixture of bacteria and moulds was detected on grass pollen. Besides Gram‐negative bacteria that are known to favour Th1‐directed immune responses, moulds were identified as being sources of allergens themselves. Herein, we focused on Gram‐positive bacteria that were found in high numbers, e.g. Bacillus cereus and Bacillus subtilis. Contact of immature dendritic cells (DC) from grass pollen allergic donors with supernatants of homogenized Gram‐positive bacteria induced maturation of DC as measured by up‐regulation of CD80, CD83 and CD86 and by enhanced production of IL‐6, IL‐12p40 and TNF‐α, which was less pronounced compared with effects induced by lipopolysaccharide (LPS). Consequently, stimulation of autologous CD4+ T cells with supernatants of homogenized Gram‐positive bacteria plus grass pollen allergen‐pulsed DC led to an enhanced proliferation and production of IL‐4, IL‐13, IL‐10, IL‐17, IL‐22 and IFN‐γ production compared with T cells that were stimulated with allergen‐pulsed immature DC alone, whereas production of the transcription factor for regulatory T cells FoxP3 was not significantly affected.Conclusions and Clinical RelevanceThese data indicate that grass pollen is colonized by several microorganisms that influence the immune response differently. Similar to LPS, supernatants of homogenized Gram‐positive bacteria may serve as adjuvants by augmenting DC maturation and inflammatory Th1, Th2 and Th17 responses helping to initiate allergic immune responses.