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. 2015 Feb 1;194(3):1057-1061.
doi: 10.4049/jimmunol.1400169. Epub 2014 Dec 17.

Stable interactions and sustained TCR signaling characterize thymocyte-thymocyte interactions that support negative selection

Heather J Melichar #  1 , Jenny O Ross #  1 , Kayleigh T Taylor  1 , Ellen A Robey  1
Affiliations

Stable interactions and sustained TCR signaling characterize thymocyte-thymocyte interactions that support negative selection

Heather J Melichar et al. J Immunol. .

Abstract

Negative selection is one of the primary mechanisms that render T cells tolerant to self. Thymic dendritic cells play an important role in negative selection, in line with their ability to induce migratory arrest and sustained TCR signals. Thymocytes themselves display self-peptide/MHC class I complexes, and although there is evidence that they can support clonal deletion, it is not clear whether they do so directly via stable cell-cell contacts and sustained TCR signals. In this study, we show that murine thymocytes can support surprisingly efficient negative selection of Ag-specific thymocytes. Furthermore, we observe that agonist-dependent thymocyte-thymocyte interactions occurred as stable, motile conjugates led by the peptide-presenting thymocyte and in which the trailing peptide-specific thymocyte exhibited persistent elevations in intracellular calcium concentration. These data confirm that self-Ag presentation by thymocytes is an additional mechanism to ensure T cell tolerance and further strengthen the correlation between stable cellular contacts, sustained TCR signals, and efficient negative selection.

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Figures

Figure 1
Figure 1. Thymocytes bearing agonist peptide efficiently support negative selection
Preselection cell proliferation dye eFluor670 labeled OT1tg DP thymocytes were mixed 1:1 with cell proliferation dye eFluor450 labeled, β2m−/− thymocyte population as an internal reference, overlaid on β2m−/− thymic slices, and allowed to migrate into the tissue for 2 hours. Thymic slices were washed, and unlabeled WT or β2m−/− thymocytes pre-loaded with or without 1nM OVA peptide were added. After an additional 2 hours, thymic slices were washed, and thymocytes were harvested at either 3 or 24 hours after the addition of the peptide-loaded thymocyte population. (A) Flow cytometry gating strategy to distinguish β2m−/− thymocyte internal reference cells versus OT1tg DP thymocytes from endogenous cells. (B) Representative flow cytometry plots of CD24 and CD69 on live, OT1tg DP thymocytes 3 hours after the addition of WT thymocytes with or without OVA peptide. (C) Representative flow cytometry plots of live, OT1tg DP thymocytes 24 hours after the addition of WT thymocytes with or without OVA peptide. Flow cytometry plots are representative of triplicate samples from one experiment of at least two. (D) Normalized ratio of live, OT1tg DP cells to internal control population 24 hours after addition of WT or β2m−/− thymocytes with or without OVA peptide. Each dot represents an individual thymic slice, and lines represent the mean. Data from one representative experiment of at least two. * indicates p < 0.05. ns, not significant (E) Representative flow cytometry plots of thymocyte preparations prior to addition to thymic slices. CD11c and MHC II on live, Lin− (CD4, CD3, TCRβ, CD19, NK1.1) from indicated populations.
Figure 2
Figure 2. Thymocytes engage in prolonged, stable interactions with thymocytes presenting negative selecting peptide
(A) Pre-selection OT1tg DP thymocyte (yellow) were overlaid on β2m−/− thymic slices in the presence of WT OVA-presenting thymocytes (aqua) and imaged by two-photon microscopy. Images were recorded in the cortex as identified by the density of OT1 thymocytes and the proximity to the thymic capsule. Representative images from a time series at the times indicated are shown. Top panels, fluorescent images. Bottom panels, lines indicate cell tracks for the duration of the movie with spots overlaid to indicate the location of each cell at the time points indicated above the top panels. (B) Time line indicating the duration of thymocyte thymocyte contacts of 13 interacting OT1tg thymocytes identified out of 265 OT1 cell tracks, in 3 imaging runs.
Figure 3
Figure 3. Thymocytes presenting negative selecting peptide support low, sustained TCR signals
(A) The frequency of time points plotted against the [Ca2+i] ratio of Indo-1–labeled preselection OT1tg DP thymocytes incubated on β2m−/− thymic slices in the presence of WT thymocytes alone or loaded with 1nM OVA peptide from two independent imaging runs. “Interacting” values are time points that were part of a thymocyte-thymocyte contact, and derived from ≥ 5 tracked OT1tg thymocytes. “Non-interacting” were values from all OT1tg thymocytes from the same run that were not in a conjugate, and that had a similar overall fluorescence value to the “interacting” tracks. (B) [Ca2+i] ratios over time from individual representative interacting OT1tg thymocyte tracks. Track labels correspond to those in Fig. 2B. Line indicates average [Ca2+i] ratio of non-interacting cells with comparable fluorescence intensity in the unbound Indo-1 channel of the interacting cell. For track I, arrow indicates the timepoint in which the initial thymocyte-thymocyte contact occurred.
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