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. 2018 May 16;9:1066. doi: 10.3389/fimmu.2018.01066

Figure 4.

Figure 4

Intracerebroventricularly (ICV)-injected, resting ovalbumin (OVA)-specific CD4 T cells undergo proliferation within the choroid plexus (CP) in a cerebrospinal fluid-antigen-dependent manner. Interferon gamma was injected intra-cisterna magna to wild-type mice, either alone or together with OVA323–339 or MOG35–55 (as a control peptide). After 24 h, the mice were ICV-injected with carboxyfluorescein succinimidyl ester (CFSE)-labeled, resting OVA-specific Th1 cells, either without a peptide (Control; n = 5), with OVA323–339 (OVA; n = 7), or with MOG35–55 [myelin oligodendrocyte glycoprotein (MOG); n = 4]. At 3-day post-injection, the lateral ventricle (LV) CPs of these mice were analyzed by flow cytometry and immunohistochemistry (IHC). (A) Flow cytometry of isolated CPs. The cellular fraction gated on mononuclear cells shows CD45+CD4+ and CFSE+ T cells. (B–F) IHC images of LV CPs obtained from the control and from the OVA-injected mice. (B) Representative brain sections of OVA-injected and control mice, immunolabeled with anti-intercellular adhesion molecule 1 (ICAM-1) (green). The graph shows fold change in ICAM-1 expression in the LV CPs of OVA-injected mice, normalized to the control mice. Scale bars represent 50 µm. (C) Representative brain sections of OVA-injected and control mice, immunolabeled with anti-CD4 (green), anti-vascular cell adhesion molecule 1 (VCAM-1) (blue), and a DAPI counterstained (gray). Scale bars represent 200 µm (top left and middle left panels), 50 µm (top right and middle right panels), and 10 µm (bottom left panel). The bottom right panel shows a 3D reconstruction of z-sections (25.9 µm overall, 0.7 µm/slice) of the framed area. (D–F) Analyses of the interactions between T cells and myeloid cells, and the proliferation of T cells within the CP. (D) Representative brain sections of OVA-injected mice, immunolabeled with anti-CD4 (green), anti-Iba-1 (red), and a TO-PRO-3 nuclear counterstain (blue). The yellow arrows indicate co-localization of CD4 and Iba-1. The right panel shows a 3D reconstruction of z-sections (9.5 µm overall, 0.5 µm/slice) of the framed area. Scale bars represent 50 µm (left) and 10 µm (middle). (E) Representative brain sections of OVA-injected mice immunolabeled with anti-Ki-67 (red) and anti-CD4 (blue). The yellow arrows indicate proliferating CD4 T cells in the CP. The right panel shows a 3D reconstruction of z-sections (22 µm overall, 2 µm/slice) of the framed area. Scale bars represent 50 µm (left) and 10 µm (right). (F) Expression pattern of CFSE and Ki-67 in CD4+ T cells, which were detected in three 40-µm brain sections of a single LV CP from a mouse injected with OVA and with OVA-specific T cells. (A,B) Each symbol represents one LV CP from an individual mouse. Bars represent means ± SEM. *P < 0.05, **P < 0.01 [(A) one-way ANOVA, (B) unpaired t test].