Abstract
BackgroundTwo-photon laser scanning microscopy (TPLSM) has become a powerful tool in the visualization of immune cell dynamics and cellular communication within the complex biological networks of the inflamed central nervous system (CNS). Whereas many previous studies mainly focused on the role of effector or effector memory T cells, the role of naïve T cells as possible key players in immune regulation directly in the CNS is still highly debated.MethodsWe applied ex vivo and intravital TPLSM to investigate migratory pathways of naïve T cells in the inflamed and non-inflamed CNS. MACS-sorted naïve CD4+ T cells were either applied on healthy CNS slices or intravenously injected into RAG1 -/- mice, which were affected by experimental autoimmune encephalomyelitis (EAE). We further checked for the generation of second harmonic generation (SHG) signals produced by extracellular matrix (ECM) structures.ResultsBy applying TPLSM on living brain slices we could show that the migratory capacity of activated CD4+ T cells is not strongly influenced by antigen specificity and is independent of regulatory or effector T cell phenotype. Naïve T cells, however, cannot find sufficient migratory signals in healthy, non-inflamed CNS parenchyma since they only showed stationary behaviour in this context. This is in contrast to the high motility of naïve CD4+ T cells in lymphoid organs. We observed a highly motile migration pattern for naïve T cells as compared to effector CD4+ T cells in inflamed brain tissue of living EAE-affected mice. Interestingly, in the inflamed CNS we could detect reticular structures by their SHG signal which partially co-localises with naïve CD4+ T cell tracks.ConclusionsThe activation status rather than antigen specificity or regulatory phenotype is the central requirement for CD4+ T cell migration within healthy CNS tissue. However, under inflammatory conditions naïve CD4+ T cells can get access to CNS parenchyma and partially migrate along inflammation-induced extracellular SHG structures, which are similar to those seen in lymphoid organs. These SHG structures apparently provide essential migratory signals for naïve CD4+ T cells within the diseased CNS.
Highlights
Two-photon laser scanning microscopy (TPLSM) has become a powerful tool in the visualization of immune cell dynamics and cellular communication within the complex biological networks of the inflamed central nervous system (CNS)
Using TPLSM, we previously showed that activated CD4+ effector T cells are attracted to the CNS’s perivascular space and reveal a CXCR4 dependent and vesselassociated migration pattern, suggesting this compartment is highly relevant for autoimmunity and immunoregulation [3,7]
Intravital imaging of the inflamed CNS of living mice reveals distinct migration pattern of naïve T lymphocytes To observe naïve T cell migration under inflammatory circumstances in the CNS, we first performed flow cytometry analysis on brain-derived immune cells after transfer of naïve T cells, which are characterized by high CD62L and low CD69, CD25 and CD44 expression (Figure 1A), into EAE affected Rag 1-/- mice
Summary
Two-photon laser scanning microscopy (TPLSM) has become a powerful tool in the visualization of immune cell dynamics and cellular communication within the complex biological networks of the inflamed central nervous system (CNS). In the last 10 years, two-photon laser scanning microscopy (TPLSM) has revealed the dynamic nature of immune cells within living lymphoid and target organs [1,2,3,4,5,6]. This has led to a better understanding of. Flow cytometry experiments showed that naïve T cells can be found in the healthy, non-inflamed CNS [8,9] This is the case for other non-lymphoid tissues including the pancreas, intestine, lung, liver, kidney, skin and testis, where it is thought that this circulation is part of the normal migratory behaviour of naïve T cells [8]. During CNS- inflammation adhesion ligands (i.e. ICAM-1 and VCAM) facilitate T cell recruitment to the CNS [10,11,12], and they only get activated when they encounter their antigen in the CNS [13]
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