Figure 5.

Increased neuronal membrane conductance during OT-I T cell–neuron interaction is attributable to incorporation of perforin into the neuronal surface membrane. A, Flow cytometry of splenocytes from RAG-1^−/− OT-I mice deficient for granzyme B (gran B^−/−; top row) or perforin (perf^−/−; bottom row) before (left column) and after (right column) in vitro stimulation with OVA and IL-2. Virtually all cells were CD8⁺ after 5 d of in vitro stimulation. B, Immunocytochemistry using specific antibodies raised against granzyme B (red, gran B) and perforin (green, perf). Cell nuclei were counterstained with DAPI (blue). Scale bar, 10 μm. C, IFN-γ production during CD3/CD28 bead stimulation of OT-I T cells and OT-I T cells deficient for granzyme B (gran B^−/−) or perforin (perf^−/−). D, Proliferation after CD3/CD28 bead stimulation of OT-I cells and OT-I cells deficient for granzyme B (granB^−/−) or perforin (perf^−/−) as indicated by [³H]thymidine incorporation. E, Bar graph representation of the alteration of R_M after establishing cell–cell contact between WT OT-I cells, OT-I cells deficient for granzyme B cluster (gran B^−/−), or perforin (perf^−/−) and hippocampal neurons. F, Representative Ca²⁺ imaging traces of Ca²⁺ signals recorded after establishing a cell–cell contact between WT OT-I T cells (n = 8) and OT-I (perf^−/−) T cells (n = 8) and neurons in the presence of OVA peptide detected at the soma. Scale bars like in Fig. 4B–D. Error bars represent mean ± SEM. **p < 0.05; ns, not significant.