Gamma delta T cells from tolerized alpha beta T cell receptor (TCR)-deficient mice inhibit contact sensitivity-effector T cells in vivo, and their interferon-gamma production in vitro

Abstract

Contact sensitivity (CS) responses to reactive hapten Ag, such as picryl chloride (PCl) or oxazolone (OX), are classical examples of T cell-mediated immune responses in vivo that are clearly subject to multifaceted regulation. There is abundant evidence that downregulation of CS may be mediated by T cells exposed to high doses of Ag. This is termed high dose Ag tolerance. To clarify the T cell types that effect CS responses and mediate their downregulation, we have undertaken studies of CS in mice congenitally deficient in specific subsets of lymphocytes. The first such studies, using alpha beta T cell-deficient (TCR alpha -/-) mice, are presented here. The results clearly show that TCR alpha -/- mice cannot mount CS, implicating alpha beta T cells as the critical CS-effector cells. However, TCR alpha -/- mice can, after high dose tolerance, downregulate alpha +/+ CS-effector T cells adoptively transferred into them. By mixing ex vivo and then adoptive cell transfers in vivo, the active downregulatory cells in tolerized alpha -/- mice are shown to include gamma delta TCR+ cells that also can downregulate interferon-gamma production by the targeted CS-effector cells in vitro. Downregulation by gamma delta cells showed specificity for hapten, but was not restricted by the MHC. Together, these findings establish that gamma delta T cells cannot fulfill CS-effector functions performed by alpha beta T cells, but may fulfill an Ag-specific downregulatory role that may be directly comparable to reports of Ag-specific downregulation of IgE antibody responses by gamma delta T cells. Comparisons are likewise considered with downregulation by gamma delta T cells occurring in immune responses to pathogens, tumors, and allografts, and in systemic autoimmunity.

Figure 3

γδ T cells are responsible for CS downregulatory activity of high dose Ag tolerized TCRα^−/− mice. In experiments 1 and 2, spleen cells (1.5 × 10⁸) from TNBSA intravenous tolerized 129/J (group B), or TCRα^−/− animals (group E), were separated into γδ TCR⁺ (groups C and F), and γδ TCR⁻ (groups D and G) subpopulations by anti-γδ TCR mAb treatment in solution, and then incubation with anti–hamster Ig-coated para magnetic beads, and subsequently separated in vitro by exposure to a magnet. In experiment 3, similar cell fractionation used α^+/+ BALB/c and their α^−/− H2^d counterparts. Immune CS-effector cells (7 × 10⁷) were incubated for 30 min at 37°C with downregulatory, separated, magnetic bead positive cells (γδ TCR⁺) (groups C and F), or magnetic bead negative (γδ TCR⁻) cells (groups D and G), that were from either TNBSA-tolerized 129/J or BALB/c mice (groups C, D), or TNBSAtolerized TCRα^−/− animals (groups F, G). Cell mixtures were then washed and injected intraperitoneally into naive recipients. As a positive control, CS-effector cells incubated without regulatory cells were transferred intraperitoneally (group A). The next day, animals were challenged on the ears with 0.8% PCl in olive oil and the increment in ear swelling was determined 24 h later. Statistical significance: group A vs B and D, P <0.01; group A vs group E, P <0.05; and group A vs group F, P <0.001.

Figure 5

Lack of MHC restriction of γδ downregulatory cells from TNBSA-tolerized TCRα^−/− mice. Magnetic bead positive (γδ TCR⁺) cells from TNBSA-tolerized α^−/− mice were incubated with 7 x 10⁷ cells from MHC compatible 129/J H2^b,d, or MHC incompatible (CBA/J, H2^k) mice for 30 min at 37°C (groups B and D). As a positive control, we used immune cells from 129/J or CBA/J mice that were incubated without regulatory cells in medium alone (groups A and C). Then cells were washed and injected intraperitoneally into recipients that were syngeneic with the immune cells, and were skin tested for CS responsiveness. Statistical significance: group A vs B, and group C vs D, P <0.001.

Figure 6

Phenotype (a) and dose response of γδ downregulatory cells derived from TNBSA-tolerized α^−/− mice (b). (a) Phenotype: To determine phenotype of downregulatory cells, spleen cells from TNBSA-tolerized α^−/− mice were incubated with medium alone, or with mAb (antiCD4 or anti-CD8) for 40 min on ice. Cells were washed and incubated with rabbit complement for 45 min at 37°C, and were washed and resuspended in PBS. 7 × 10⁷ CS-effector cells were incubated with medium alone (group A, positive control), with 5 × 10⁷ untreated tolerized cells (group B), or with 5 × 10⁷ anti-CD4 + C treated tolerized cells, (group C), or with 5 × 10⁷ anti-CD8 + C treated tolerized cells, (group D). Statistical significance: group A vs B, and group A vs C P <0.001, and group A vs D P <0.01. (b)-Dose response: 7 × 10⁷ immune CS effector cells were incubated with medium alone (group A, positive controls), or with 10-fold decreasing dilutions (2.5 × 10⁵, 2.5 × 10⁴, or 2.5 × 10³ cells) of magnetic bead positive (γδ TCR⁺) downregulatory cells from α^−/− TNBSA-tolerized mice, for 30 min at 37°C (groups B–D). After incubation, the cell mixtures were washed and injected into recipients that were challenged on the ears, and measured for CS responses at 24 h. Statistical significance: group A vs B and C, P <0.001, group A vs D, P <0.01.

Figure 6

Phenotype (a) and dose response of γδ downregulatory cells derived from TNBSA-tolerized α^−/− mice (b). (a) Phenotype: To determine phenotype of downregulatory cells, spleen cells from TNBSA-tolerized α^−/− mice were incubated with medium alone, or with mAb (antiCD4 or anti-CD8) for 40 min on ice. Cells were washed and incubated with rabbit complement for 45 min at 37°C, and were washed and resuspended in PBS. 7 × 10⁷ CS-effector cells were incubated with medium alone (group A, positive control), with 5 × 10⁷ untreated tolerized cells (group B), or with 5 × 10⁷ anti-CD4 + C treated tolerized cells, (group C), or with 5 × 10⁷ anti-CD8 + C treated tolerized cells, (group D). Statistical significance: group A vs B, and group A vs C P <0.001, and group A vs D P <0.01. (b)-Dose response: 7 × 10⁷ immune CS effector cells were incubated with medium alone (group A, positive controls), or with 10-fold decreasing dilutions (2.5 × 10⁵, 2.5 × 10⁴, or 2.5 × 10³ cells) of magnetic bead positive (γδ TCR⁺) downregulatory cells from α^−/− TNBSA-tolerized mice, for 30 min at 37°C (groups B–D). After incubation, the cell mixtures were washed and injected into recipients that were challenged on the ears, and measured for CS responses at 24 h. Statistical significance: group A vs B and C, P <0.001, group A vs D, P <0.01.

Figure 1

Actively sensitized TCRα knock out mice (α^−/−) have no classical late 24 h cutaneous CS responses, but can serve as adoptive cell transfer recipients. TCRα^+/+ 129/J (group A) or TCRα^−/− (group B) mice, were contact sensitized with 0.15 ml of 5% PCl skin painting. 4 d later, animals were challenged on the ears with 0.8% PCl, and CS responses were determined 24 h later by measuring increases in ear thickness with an engineer's micrometer. Results were expressed as U × 0.01 mm ± SE. Background ear swelling in PCl challenged controls that were not sensitized, were subtracted from experimental groups to give net ear swelling, as is shown. BDF₁ (group C) α^+/+ immune cells from PCl contact sensitized donors were transferred intravenously to BDF₁ recipients, or to TCRα^−/− H2^b recipients (group D) that were ear challenged similarly, and then, 24 h ear swelling responses were determined.

Figure 2

High dose Ag tolerance of TCRα^−/− mice induces cells that downregulate CS and generate an inhibitory environment. As an assay for generation of downregulatory cells, CS-effector cells (7 × 10⁷) from PCl contact sensitized 129/J mice (groups A–D) were incubated for 30 min at 37°C, alone (group A, positive controls), or with 5 × 10⁷ putative regulatory cells induced by two intravenous injections of TNBSA in TCRα^+/+ 129/J (group B), TCRα^+/− (group C), or TCRα^−/− (group D) mice. The cell mixture was then washed and transferred intravenously into naive syngeneic 129/J recipients that immediately were challenged on the ears with 0.8% PCl. Groups B–D were significantly different (P <0.001) from control group A. In groups E–I, from a pooled separate experiment, TCRα^+/+ BDF₁ mice or their TCRα^−/− H2^b,d counterparts were used. BDF₁ are poorly CS-reactive and thus were skin sensitized with PCl twice (days 0 and 3). On d 7, immune CS-effector lymph node and spleen cells (7 × 10⁷), from these mice were injected intravenously into normal BDF₁ mice (groups E–G), or into TCRα^−/− recipients (groups H and I). Tolerized downregulatory cells were induced in BDF₁ or TCRα^−/− mice by two intravenous injections of 5 × 10⁷ TNP-conjugated TCRα^−/− spleen cells on days 0 and 3. Spleens from these tolerized mice served as a source of downregulatory cells respectively in groups F and G, in which 3.5 × 10⁷ tolerized cells were mixed and incubated with BDF₁ PCl-immune cells (7 × 10⁷), before being injected intravenously together into the recipients. In group H, recipient TCRα^−/− animals were tolerized via prior intravenous injection of TNP-conjugated spleen cells, i.e., they were tolerized by a procedure similar to inducing regulatory cells used in the mixing and adoptive transfer experiments (groups E–G). Statistical significance: group E vs F, I and H, P <0.005. Each group consisted of three animals. In group I, BDF₁ CS-immune cells were transferred successfully into untreated TCRα^−/− mice.

Figure 4

(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.

Figure 4

(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.

Figure 7

(a) γδ T cells in spleens of TNBSA intravenously tolerized α^−/− mice Ag-specifically downregulate IFN-γ production by CS-effector T cells. 2 × 10⁵ TNP-immune CS-effector cells from α^+/+ mice were cultured in vitro in the presence of TNP-conjugated normal spleen cells as APC, either with or without added regulatory cells (group A), or with regulatory cells of two different specificities: either TNBSA-tolerized or OXtolerized. These regulatory cells were either from α^+/+ normal mice (groups B and C), or were harvested from TNBSA-tolerized or OX-tolerized α^−/− mice (groups E and F, respectively). At the end of 48 h, supernatants from the individual microwell cultures were harvested and assayed subsequently for IFN-γ content with a double mAb capture ELISA immunoassay. Statistics: group A vs B, P <0.004; group C vs A, P <0.01; group E vs D, P <0.004; and group F vs D, P <0.02. (b) Phenotype of in vitro γδ down regulatory cells: 2 × 10⁵ lymph node CS-effector cells from PCl-sensitized donors were cultured in individual wells of flat-bottomed wells of microtrays in quadruplicate with TNP- or OX-conjugated syngeneic spleen cells as APC, either alone (groups A and D), or with regulatory cell subpopulations from TNBSA-tolerized or OX-tolerized donors (group B and groups C, E, and F). These regulatory cells came from either normal α^+/+ mice (groups B and C), or from matched TCRα knockout α^−/− mice. Prior to addition to microcultures, these regulatory cells were each separated by immunomagnetic bead techniques, using anti-γδ TCR mAb, into γδenriched cells (γδ⁺, left), or γδ-depleted cells (γδ⁻, right). After 48 h culture of the various cell mixtures, supernatants were harvested from individual wells and assayed subsequently using a two mAb capture ELISA immunoassay for IFN-γ content. Statistics: group A vs B (left), NS; group A vs B (right), P <0.001; group A vs C (right), P <0.001; group D vs E (left), P <0.002; group D vs F (left), NS; group D vs E (right), NS.

Figure 7

(a) γδ T cells in spleens of TNBSA intravenously tolerized α^−/− mice Ag-specifically downregulate IFN-γ production by CS-effector T cells. 2 × 10⁵ TNP-immune CS-effector cells from α^+/+ mice were cultured in vitro in the presence of TNP-conjugated normal spleen cells as APC, either with or without added regulatory cells (group A), or with regulatory cells of two different specificities: either TNBSA-tolerized or OXtolerized. These regulatory cells were either from α^+/+ normal mice (groups B and C), or were harvested from TNBSA-tolerized or OX-tolerized α^−/− mice (groups E and F, respectively). At the end of 48 h, supernatants from the individual microwell cultures were harvested and assayed subsequently for IFN-γ content with a double mAb capture ELISA immunoassay. Statistics: group A vs B, P <0.004; group C vs A, P <0.01; group E vs D, P <0.004; and group F vs D, P <0.02. (b) Phenotype of in vitro γδ down regulatory cells: 2 × 10⁵ lymph node CS-effector cells from PCl-sensitized donors were cultured in individual wells of flat-bottomed wells of microtrays in quadruplicate with TNP- or OX-conjugated syngeneic spleen cells as APC, either alone (groups A and D), or with regulatory cell subpopulations from TNBSA-tolerized or OX-tolerized donors (group B and groups C, E, and F). These regulatory cells came from either normal α^+/+ mice (groups B and C), or from matched TCRα knockout α^−/− mice. Prior to addition to microcultures, these regulatory cells were each separated by immunomagnetic bead techniques, using anti-γδ TCR mAb, into γδenriched cells (γδ⁺, left), or γδ-depleted cells (γδ⁻, right). After 48 h culture of the various cell mixtures, supernatants were harvested from individual wells and assayed subsequently using a two mAb capture ELISA immunoassay for IFN-γ content. Statistics: group A vs B (left), NS; group A vs B (right), P <0.001; group A vs C (right), P <0.001; group D vs E (left), P <0.002; group D vs F (left), NS; group D vs E (right), NS.