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. 1996 Dec 1;184(6):2129–2140. doi: 10.1084/jem.184.6.2129

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(a) Reciprocal Ag specificity of splenic γδ⁺ regulatory cells from TNP or OX-tolerized α^−/− mice that act on TNP versus OX-immune CS-effector T cells. BDF₁ mice were used as donors and recipients of PCl or OX immune cells, while their H2^b,d TCRα^−/− counterparts were used as donors of tolerized regulatory cells. To tolerize spleens of TCRα^−/− mice, normal α^−/− spleen cells were conjugated in vitro with TNP or OX, and 5 × 10⁷ hapten-conjugated self cells were injected intravenously into α^−/− mice on days 0 and 3, and then spleen cell suspensions from these tolerized recipients were prepared on day 7 as putative regulatory cells. BDF₁ CS-effector cell donors were contact skin-sensitized with TNP (via PCl painting) (groups A–C), or with OX (groups D–F), on days 0 and 3, and then spleen and lymph node cells were transferred into BDF₁ recipients on day 7, either alone (7 × 10⁷ cells) (groups A and D), or mixed with Ag homologous downregulatory α^−/− cells (groups C and E), or Ag heterologous downregulatory cells (5 × 10⁷) from TCRα^−/− mice (groups B and F). Adoptive cell recipients that were MHC homologous to CS-effector cells that were cotransferred, were challenged on the ears with TNP or OX hapten, and ear swelling measurements were made 24 h later. Each group consisted of three mice. Statistical significance: group A vs C, P <0.001; and group D vs E, P < 0.002; group D vs F NS. (b) Antigen-specificity of tolerance in α^−/− mice tested as recipients of α^+/+ CS-effector cells. To confirm Ag-specificity of tolerized α^−/− mice we used an assay that did not involve transfers and manipulation of the putative regulatory cells. α^−/− mice were tolerized by intravenously injection of TNBSA or OX-MRBC before serving as recipients for transfer of CS-effector cells from normal BALB/c mice that were of homologous or unrelated Ag (TNP vs OX) specificity. The experiments included groups of positive and negative controls. Mice were then challenged immediately on the ears with homologous hapten (TNP or OX), and then tested for CS ear swelling 24 and 48 h later. Statistical analysis at 24 h using the two tailed Student's t test showed: group B vs A, P <0.01; group B vs C, P <0.02; group E vs D, P <0.01; group E vs F, P <0.01. For 48 h: group B vs A, P <0.02; group B vs C, P <0.01; group E vs D, P <0.001; and group E vs F, P <0.01.

(a) Reciprocal Ag specificity of splenic γδ⁺ regulatory cells from TNP or OX-tolerized α^−/− mice that act on TNP versus OX-immune CS-effector T cells. BDF₁ mice were used as donors and recipients of PCl or OX immune cells, while their H2^b,d TCRα^−/− counterparts were used as donors of tolerized regulatory cells. To tolerize spleens of TCRα^−/− mice, normal α^−/− spleen cells were conjugated in vitro with TNP or OX, and 5 × 10⁷ hapten-conjugated self cells were injected intravenously into α^−/− mice on days 0 and 3, and then spleen cell suspensions from these tolerized recipients were prepared on day 7 as putative regulatory cells. BDF₁ CS-effector cell donors were contact skin-sensitized with TNP (via PCl painting) (groups A–C), or with OX (groups D–F), on days 0 and 3, and then spleen and lymph node cells were transferred into BDF₁ recipients on day 7, either alone (7 × 10⁷ cells) (groups A and D), or mixed with Ag homologous downregulatory α^−/− cells (groups C and E), or Ag heterologous downregulatory cells (5 × 10⁷) from TCRα^−/− mice (groups B and F). Adoptive cell recipients that were MHC homologous to CS-effector cells that were cotransferred, were challenged on the ears with TNP or OX hapten, and ear swelling measurements were made 24 h later. Each group consisted of three mice. Statistical significance: group A vs C, P <0.001; and group D vs E, P < 0.002; group D vs F NS. (b) Antigen-specificity of tolerance in α^−/− mice tested as recipients of α^+/+ CS-effector cells. To confirm Ag-specificity of tolerized α^−/− mice we used an assay that did not involve transfers and manipulation of the putative regulatory cells. α^−/− mice were tolerized by intravenously injection of TNBSA or OX-MRBC before serving as recipients for transfer of CS-effector cells from normal BALB/c mice that were of homologous or unrelated Ag (TNP vs OX) specificity. The experiments included groups of positive and negative controls. Mice were then challenged immediately on the ears with homologous hapten (TNP or OX), and then tested for CS ear swelling 24 and 48 h later. Statistical analysis at 24 h using the two tailed Student's t test showed: group B vs A, P <0.01; group B vs C, P <0.02; group E vs D, P <0.01; group E vs F, P <0.01. For 48 h: group B vs A, P <0.02; group B vs C, P <0.01; group E vs D, P <0.001; and group E vs F, P <0.01.