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. 2022 Jul 6;13:931630. doi: 10.3389/fimmu.2022.931630

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Copyright © 2022 Lelliott, Ramsbottom, Dowling, Shembrey, Noori, Kearney, Michie, Parish, Jordan, Baxter, Young, Brennan and Oliaro

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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NKG7 protein is localized to late endosomes and polarizes to the immune synapse upon target recognition. (A) Confocal immunofluorescence microscopy of Nkg7^+/+ CD8+ T cells showing co-localization of NKG7 (magenta) with Lamp-1 and Rab-7 (green; middle panels), but not EEA1 or GzmB (green; far left and right panels). Both Costes Pearson’s Correlation Coefficient and Manders Correlation Coefficient, were used to quantify co-localization (left); biological replicates (right). Comparisons made by one-way ANOVA, each dot represents an individual cell analyzed (n = 47-133) pooled from 3 independent experiments (average shown on right) (B) The percentage of NKG7 (green) present at the MTOC (magenta) for synapsed and un-synapsed Nkg7^+/+ CD8+ T cells, with synapse determined by polarisation of the MTOC to the interface of the two cells. Anti-CD3/28-coated P815 target cells were labelled with cell trace violet (CTV) and co-cultured with Nkg7^+/+ CD8+ T cells for 45 mins. Comparisons made by unpaired t test. Each dot represents an individual cell conjugate analyzed (n = 30-51) pooled from 3 independent experiments (average shown below) (C) The percentage of granzyme B (GzmB; green) present at the immune synapse (MTOC; red) in Nkg7^+/+ and Nkg7^-/- CD8+ T cells, during co-culture with target cells as described in (B), unpaired t test. Each dot represents an individual cell conjugate analyzed (n = 23-28) pooled from 3 independent experiments (average shown below) * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant.