Effect of CD4⁻ iNKT cells on MLR. (A) αGC-expanded, highly purified CD4⁻ iNKT cells from a normal donor (R86) were placed in MLR comprising autologous CD3⁺ T cells (responders; R) against allogeneic irradiated PBMC (stimulators; S) at iNKT/R/S ratio of 10:1:1 and compared with R/S (baseline) and autologous (self) MLR. Cell proliferation was measured by ³H-thymidine incorporation 96 hours later. Bars represent means and error bars represent SEMs of triplicate assays. (B) In a series of MLRs involving 8 different responders, each placed against a panel of 3-4 allogeneic stimulators, the addition of autologous CD4⁻ iNKT cells resulted in a 59% ± 8% (P = .0048; 1 sample z test) decrease in proliferation compared with baseline MLR. (C) IFN-γ secretion was significantly decreased on the addition of CD4⁻ iNKT cells to the baseline MLR of R86 against the same panel of stimulators as in panel A. Data are shown as mean ± SEM of triplicate assays. (D) Dose-dependent inhibition of the MLR by CD4⁻ iNKT cells. Responder 75 (R75) CD4⁻ NKT cells were added in MLR at different iNKT/R ratios as shown. Data are shown as mean ± SEM of triplicate assays. (E) Contact-dependent inhibition of the MLR by CD4⁻ iNKT cells. MLRs of R75 versus 2 stimulators were set up in a transwell coculture system as shown at the bottom and described in “Methods.” Contact of CD4⁻ NKT cells with the responder and stimulator cells suppressed the MLR; when CD4⁻ iNKT cells were separated from the MLR by the transwell membrane, cell proliferation was almost completely restored to the levels of the baseline MLR. Data are shown as mean ± SEM of triplicate assays. (F) CD4⁻ iNKT cells are cytotoxic against allogeneic myeloid DCs. CD4⁻ iNKT cells were cytotoxic against allogeneic DCs in a Cr⁵¹ release assay at the iNKT/DC ratios of 1:1 and 10:1, an effect that was abrogated by the Ca²⁺ chelator EGTA. Data are shown as mean ± SEM of triplicate assays. See also supplemental Figure 6.