1 Introduction [1,2] [3,4] [5] [6,7] [8,9] [10,11] [4,12] [13] [14] Staphylococcus aureus [15] [8,11] [2] [1,2] [16] [17] [17] Human studies of antigen-specific neutrophil-T cell interactions are confounded by low frequency of antigen-specific T cells in peripheral blood, variation of MHC allele expression, which therefore impedes ideal peptide antigen identification. To overcome these complications, we have employed a murine model system. Moreover, despite the important role of the neutrophil in the pathogenesis of numerous inflammatory diseases, many of which are modelled in rodents, there is a relative paucity of information on murine Class II restricted antigen presentation. We, therefore, sought to investigate whether murine neutrophils present antigens via MHC Class II. 2 Materials and methods 2.1 Murine peritoneal neutrophil and macrophage separation [18] ® g 6 −1 l 6 −1 2.2 Murine lymph node preparation + 323–339 d [19] 6 −1 3 2.3 Assessment of cell proliferation and cytokine production 3 3 3 2.4 Hybridoma cells [20] 323–339 2.5 Flow cytometry 6 3 + 323–339 2.6 Statistical analysis Statistical analysis was performed on data as indicated in the figure legends, using Minitab software. 3 Results 3.1 Purification of murine neutrophils [18] [18] Fig. 1 Fig. 1 Fig. 1 Fig. 1 3.2 Murine neutrophils are able to present peptide antigen 323–339 d Fig. 2 323–339 Fig. 2 3.3 Neutrophils can drive naïve T cell proliferation and cytokine production 323–339 Fig. 3 323–339 323–339 Fig. 3 Fig. 3 Fig. 3 3.4 Cell contact dependence of neutrophil T cell interactions 323–339 323–339 Fig. 4 323–339 Fig. 4 Fig. 4 323–339 Fig. 4 4 Discussion Data presented here demonstrate that murine neutrophils are indeed capable of directly presenting antigen on Class II MHC and activating T cells independently of other APCs. The DO11.10-GFP hybridoma cells respond exclusively in a Class II restricted manner, and our data demonstrate that neutrophils, rather than contaminating cells, were mediating this response. At comparable cell ratios, neutrophil-mediated T cell activation was lower than that mediated by macrophages, commensurate with reduced expression of MHC Class II by neutrophils. Although neutrophils have been reported to express co-stimulatory molecules, we detected only low levels of CD40 and were unable to consistently detect CD80 or CD86 in our neutrophil preparations (in contrast to the F4/80 positive macrophages which expressed CD40, CD80 and CD86; data not shown). Thus it is possible that: co-stimulatory molecule expression below the limits of detection of methods used here is sufficient for inducing a response, that T cells are responding in a co-stimulation independent manner, or that co-stimulation has occurred via alternative molecules. 323–339 E. coli [17] [21] 323–339 [22] [15] [16] [23] Fig. 2 Fig. 3 There are overall fewer studies of murine neutrophils compared to human neutrophils, which is likely to be a reflection of the ease of acquiring human neutrophils that constitute 70% of circulating leucocytes, compared to limited quantity of murine peripheral blood available and the relative paucity of neutrophils, contributing only 20% of circulating leukocytes in rodents. These studies were only possible through use of rodent cells, as the TCR transgenic T cells absolutely control antigen specificity of response, which was central to this investigation of neutrophil-T cell interactions. However, the significantly different proportion of neutrophils in murine and human peripheral blood raises questions as to whether murine and human neutrophils perform parallel functions as is assumed. Our data document a novel, Class II restricted antigen presenting function of murine neutrophils, confirming findings in other human systems, and expanding our understanding of the role of the neutrophil in the adaptive immune response.