In vivo polarization of IFN-gamma at Kupfer and non-Kupfer immunological synapses during the clearance of virally infected brain cells - PubMed (original) (raw)

FIGURE 1

T cells establishing Kupfer-type immunological synapses display IFN-γ that is polarized toward virally infected cells. A, Two Kupfer-type immunological synapses, with the characteristic central distribution of TCR (defining the c-SMAC), and the peripheral distribution of LFA-1 (defining the p-SMAC), and which express IFN-γ are illustrated in detail. Immunological synapses were stained for IFN-γ (green) and TCR (red) (synapse 1, top two rows); or IFN-γ (green) and LFA-1 (red) (synapse 2, bottom two rows) combined with HSV1-TK (white, a marker protein expressed by adenovirally infected cells), and nuclear staining (DAPI, blue). a1, A three dimensional composite of the stack of confocal images at low power throughout the tissue section illustrating T cells (TCR, red) in close apposition with the cell body of a virally infected cell (HSV1-TK, white). b1_–_e1, High power images of the area outlined in the yellow box in a1 showing nuclei (blue, DAPI), IFN-γ (green), TCR (red), and the cell body of a virally infected cell (HSV1-TK, white). f1, Merged images of DAPI (b1), IFN-γ (c1), TCR (d1), and the infected cell (d1) illustrating the polarized distribution of IFN-γ (green) at the immunological synapse with the virally infected cell body and colocalization with the accumulation of TCR in the c-SMAC. h1, Fluorescence intensity graph of IFN-γ (green) and TCR (red) along the cell outline (white line, g1) demonstrating the colocalization of highest levels of intensity of IFN-γ and TCR along the c-SMAC of the immunological synapse. i1_–_l1, 3-D images were reconstructed along the interface (yellow line) and viewed en face (from the direction of the white arrow) to reveal polarized distribution of IFN-γ (j1, green) and c-SMAC (k1, TCR, red). l1, Merged 3-D reconstructed images showing the localization of IFN-γ to the c-SMAC outlined by TCR. a2, A three dimensional composite of the stack of confocal images at low power throughout the tissue section illustrating T cells (LFA-1, red) in close apposition with the cell body of a virally infected cell (HSV1-TK, white). b2_–_e2, High power images of the area outlined in the yellow box (a2) showing nuclei (blue, DAPI), IFN-γ (green), LFA-1 (red), and a virally infected cell (HSV1-TK, white). f2, Merged image of DAPI (b2), IFN-γ (c2), LFA-1 (d2), and the infected cell (e2) illustrating the polarized distribution of IFN-γ (green) at the center of the immunological synapse (c-SMAC) with the virally infected cell body; LFA-1 outlines the p-SMAC. h2, Fluorescence intensity graph of IFN-γ (green) and LFA-1 (red) along the cell outline (white line, g2) demonstrating the highest levels of intensity of LFA-1 were along the peripheral compartment of the immunological synapse, i.e., p-SMAC, surrounding the IFN-γ located within the c-SMAC. i2_–_l2, 3-D images were reconstructed along the interface (yellow line) and viewed en face (from the direction of the white arrow) to reveal the polarized distribution of IFN-γ (j2, green) and p-SMAC (k2, LFA-1, red). l2, Merged 3-D reconstructed images showing the localization of IFN-γ central to the p-SMAC outlined by LFA-1. Scale bar (f1) = 10 _μ_m. B shows a schematic of the method to quantitate the diameter of the various constituents of the Kupfer-type immunological synapses. The diameter of the interface (I) at mature immunological synapses was defined by the external limit of the LFA-1 ring (p-SMAC), the diameter of the c-SMAC was defined by the internal boundary of LFA-1, the diameter of TCR cluster was determined by TCR expression, and the diameter of the area occupied by IFN-γ immunoreactivity was also measured. *, p < 0.05 with respect to C, indicating that the area occupied by IFN-γ is smaller than the c-SMAC; †, p < 0.05 with respect to IFN-γ, TCR and C, indicating that c-SMAC, and the immunoreactivity of TCR and IFN-γ were smaller than the p-SMAC. No statistical difference was found between TCR and c-SMAC quantitation. C shows that the average diameter of the synaptic interface (I) was ∼8 _μ_m, the diameter of c-SMAC was ∼4 _μ_m. The IFN-γ cluster displayed a smaller diameter (∼2.6 _μ_m) significantly different from the c-SMAC. D illustrates the various morphologies adopted by the p-SMAC, defined by LFA-1 immunoreactivity. Measurements of LFA-1 (used to calculate pSMAC and c-SMAC) were taken using the confocal software, and are based on the quantification of 39 synapses; the quantification of the IFN-γ and TCR in Kupfer-type synapses is from 7 immunological synapses.