Sequential Assessment of Cell Cycle S Phase in Flow Cytometry: A Non-Isotopic Method to Measure Lymphocyte Activation In Vitro

Lymphocyte multiplication can be induced in vitro by mitogens or specific antigens, and is usually measured using isotopic methods involving tritiated thymidine. Cellular proliferation can also be analyzed by flow cytometry techniques based on cell cycle analysis through the measurement of DNA content. We applied this method to lymphocytes from 113 individuals, to evaluate lymphocyte proliferation after stimulation in vitro by a mitogen (phytohaemagglutinin, PHA) or a recall antigen (tetanus toxoid), using a kinetic approach with four points sequential measurements of the S and G2 phases over six days of culture. The proportion of cells in S phase after PHA stimulation was significantly higher than in controls overall and as early as on day three of the culture. Activation with a recall antigen significantly induced increasing S phase cell proportions up to day six. These data suggest that flow cytometric assessment of the S phase could be a useful alternative to isotopic methods measuring lymphocyte reactivity in vitro

Enhanced expression of l-selectin on peripheral blood lymphocytes from patients with IgA nephropathy

To investigate the homing characteristics of T and B lymphocytes which could explain the abnormal partition of IgA-producing cells in tonsils and bone marrow from patients with IgA nephropathy (IgAN), the expression of leucocyte adhesion molecules (CD11a, CD29, CD49d, CD62L, CD31) was assessed using flow cytometry on peripheral blood leucocytes from patients with biopsy-proven IgAN and controls. Higher proportions of T and B lymphocytes expressing higher amounts of l-selectin, as well as higher proportions of B cells expressing more CD31 were evidenced in IgAN patients. Conversely, serum levels of sCD62L were not different from controls, but significantly higher than serum levels in patients suffering from other renal diseases. We hypothesize that this over-expression of CD62L and CD31 may be involved in an enhanced efficiency of lymphoid cells homing to lymphoid tissues in this disease

Nogo-receptor 1 deficiency has no influence on immune cell repertoire or function during experimental autoimmune encephalomyelitis

The potential role of Nogo-66 Receptor 1 (NgR1) on immune cell phenotypes and their activation during neuroinflammatory diseases such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), is unclear. To further understand the function of this receptor on haematopoietically-derived cells, phenotypic and functional analyses were performed using NgR1-deficient (ngr1-/-) animals. Flow cytometry-based phenotypic analyses performed on blood, spleen, thymus, lymph nodes, bone marrow and central nervous-system (CNS)-infiltrating blood cells revealed no immunological defects in naïve ngr1-/- animals versus wild-type littermate (WTLM) controls. EAE was induced by either recombinant myelin oligodendrocyte glycoprotein (rMOG), a model in which B cells are considered to contribute pathogenically, or by MOG35-55 peptide, a B cell-independent model. We have demonstrated that in ngr1-/- mice injected with MOG35-55, a significant reduction in the severity of EAE correlated with reduced axonal damage present in the spinal cord when compared to their WTLM controls. However, despite a reduction in axonal damage observed in the CNS of ngr1-/- mice at the chronic stage of disease, no clinical differences could be attributed to a specific genotype when rMOG was used as the encephalitogen. Following MOG35-55-induction of EAE, we could not derive any major changes to the immune cell populations analyzed between ngr1-/- and WTLM mice. Collectively, these data demonstrate that NgR1 has little if any effects on the repertoire of immune cells, their activation and trafficking to the CNS