Bacterial stimulation plays an important role in modulating the allergic immune response. The aim of this study was to investigate the effects of inactivated probiotic Lactobacillus acidophilus and non-pathogenic Escherichia coli strain Nissle on the phenotype and function of T- and B-cells. Peripheral blood mononuclear cells from patients with grass-pollen allergy (n=10) and non-allergic patients (n=19) were co-stimulated with inactivated bacteria and grass-pollen allergen. Expression of CD23, CD80, CD86 and CD69 were analysed, and the intracellular production of interleukin-4 and interferon-gamma was measured by direct ex vivo flow cytometry after stimulation. Both bacteria induced a significant up-regulation of CD69 expression on T-lymphocytes (p=0.001). CD23 expression was significantly increased following stimulation with allergen (p=0.008), but reduced after stimulation with Lactobacillus and significantly reduced with E. coli plus allergen (p=0.029). CD80 expression was reduced after stimulation with Lactobacillus in the allergic group only (p=0.021). By contrast, CD86 expression was significantly increased after stimulation with Lactobacillus (p=0.049) and distinctly increased with E. coli in both groups (p=0.001). The cytokine patterns of CD69-positive T-lymphocytes from allergic patients showed a TH2-dominated response after allergen stimulation (interferon-gamma/interleukin-4-ratio 0.2), directed into a T-helper1-like response by stimulation with both types of bacteria (interferon-gamma/interleukin-4-ratios 1.5-2.0 in both groups). These data show that both types of bacteria modulate the allergic immune response by the alteration of CD23 and co-stimulatory molecule expression. Regarding cytokine production, the data suggest a differential response to both bacteria depending on the atopic state, but a clear promotion of T-helper1-dominated response in allergic donors.

Cellular inflammation of the nasal mucosa demonstrates a local immune response which plays an important role in allergic rhinitis. The aim of the present study was to characterize nasal mucosal lymphocytes regarding their activation and differentiation state by direct ex vivo flowcytometric analysis. Lymphocytes from the inferior turbinates were isolated by a mechanical method of preparation and, for comparison, from peripheral blood by Ficoll gradient centrifugation. Patients suffering from rhinitis or difficulty in nasal breathing were divided into an allergic (pollen-allergy, n = 13) and non-allergic group (n = 24). Expression of different T- and B-cell markers was determined by flowcytometric analysis. CD4+ T-cells from the nasal mucosa exhibited a memory phenotype (CD45RO+, 97%), were highly activated (CD69+, 43-73%), and showed low expression of the cutaneous lymphocyte antigen (CLA+, 5%). Nasal CD20+ B-lymphocytes expressed significantly higher levels of mIgE and lower levels of CD23 and CD80 than peripheral B-cells. Subsets of CD80+ (4%) and CD86+ (6%) CD20+ B-lymphocytes were identified in the nasal mucosa. No significant differences between allergic and non-allergic individuals were determined. As expected, the data show profound phenotypical differences between circulating peripheral blood and nasal mucosal lymphocytes. Activated memory lymphocytes are present in the nasal mucosa from allergic, but also non-allergic patients and may indicate to a significant role of a local inflammatory state without systemic criteria for allergy. In our study, we show that direct ex vivo isolation of lymphocytes is practicable method and offers a new technique to examine the local nasal allergic immune response using a multiparametric phenotypical analysis.

Background Acute coronary syndromes (ACS) remain the most devastating clinical manifestation of cardiovascular disease, the most common cause of global mortality. ACS frequently arises from rupture of the coronary fibrous cap, in which a highly thrombogenic necrotic core is exposed to circulating blood, thereby triggering thrombus formation and impairing myocardial perfusion. While inflammation has been implicated as a key mechanism contributing to atherosclerotic plaque vulnerability and rupture, the underlying mechanisms leading to coronary plaque erosion, another form of ACS, are not well understood. In recent findings, flow cytometric (FACS) analysis of blood from the site of patients with culprit lesion plaque erosion showed significant enrichment of both CD4+ and CD8+ T-lymphocytes (+8.1% and +11.2%, respectively, both P<0.05) as well as effector molecules such as granzyme A (+22.4%), perforin (+58.8%), and granulysin (+75.4%) as compared with patients with plaque rupture culprit lesion. The proximity of eroded lesions to coronary bifurcations seen by optical coherence tomography provides further elucidations linking shear stress alterations to the occurrence of erosional ACS. Purpose We aim to further explore the underlying immune cell mechanisms of coronary plaque erosion via a novel single cell multi-omic approach, never before established from samples aspirated from the arterial sheath. Methods Patients presenting with Myocardial Infarction underwent emergent coronary angiography & percutaneous coronary intervention. Infarction was characterized as either coronary plaque rupture or erosion by optical coherence tomography, in which then blood samples were obtained by being aspirated directly along the arterial sheath. Peripheral blood mononuclear cells were isolated via density gradient within four hours of sample aspiration and cryopreserved. Samples from 24 patients were promptly thawed, washed and incubated with 137 oligonucleotide barcoded antibodies to detect surface protein expression as well as a cellular hashtag antibody. Bioinformatic integration of single cell epitope and transcriptome information was performed derived from custom pipelines. Results Our analysis found 32 unique leukocyte clusters that allowed us to rigorously compare each patients sequenced single cell data -totaling approximately 140,000 singe cells. Additionally, the discovery of T cell oligoclonality amongst certain disease patients alludes to antigen-specific activation. We also observed increased CCL4 expression in CD8+ T-lymphocytes within erosional ACS. Conclusion Our high-parametric immune cell analysis shows distinct differences in the adaptive immune system, particularly CD8+ T-lymphocytes and their effector molecules, in the pathogenesis of erosional versus ruptured ACS. Furthermore, our observations found on the oligoclonal expansion of T cell clones aids us to further elucidate underlying mechanisms of culprit lesion formation in ACS. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): DGK - German Cardiac SocietyDGF - TRR 259 Aortic Disease German Research Foundation

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