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Development of contact hypersensitivity in mice depends on the migration of Langerhans cells from the epidermis to regional lymph nodes. Since ICAM-1 and LFA-1 play important roles in leukocyte migration, we sought to determine whether in vivo administration of anti-ICAM-1 and anti-LFA-1 antibodies would inhibit contact sensitization-induced migration of epidermal Langerhans cells to regional lymph nodes. Twenty-four hours after contact sensitization of mice with FITC, a brightly FITC-stained Ia+ population of dendritic cells capable of stimulating a FITC-specific Ia-restricted T-cell hybridoma was readily detected in their draining lymph nodes. Animals treated with anti-Ia mAb, which depletes Ia+ cells in lymph nodes and spleen but not Ia+ Langerhans cells in the epidermis, had normal numbers of FITC-bearing Ia+ cells capable of stimulating the T-cell hybridoma. Dendritic lymph node cells from mice treated with anti-ICAM-1 and anti-LFA-1 mAb were devoid of brightly FITC-stained cells and cells capable of stimulating the FITC-specific T-cell hybridoma. The combination of anti-ICAM-1 and anti-LFA-1 mAb completely inhibited the induction of contact hypersensitivity to FITC. Animals treated with anti-ICAM-1 or anti-LFA-1 monoclonal antibodies alone had significantly reduced (by 79 and 36%, respectively) numbers of brightly stained cells capable of stimulating the hybridomas. These data suggest that the adhesion molecules, ICAM-1 and LFA-1, play a significant role in contact hypersensitivity-induced migration of Langerhans cells to regional lymph nodes. The immunomodulatory effects of anti-adhesion molecule antibodies in vivo may be in part due to their effects on antigen-presenting cell migration.

We produced a series of T cell hybridomas that produce IL-2 when cultured with syngeneic APC coupled to FITC or TNP. These hybridomas are hapten specific and Ia restricted. The hybridomas were used to detect hapten-bearing APC in draining lymph nodes of mice sensitized with trinitrochlorobenzene or FITC in vivo. Hapten-bearing APC capable of stimulating the hybridomas were detectable in draining lymph nodes of hapten-painted mice within 3 h after sensitization. The ability of lymph node APC to stimulate the hybridomas peaked at 24 h and declined by 48 h. The dendritic cell subpopulation was the subpopulation of cells that were found in the regional lymph nodes of hapten-painted animals that were capable of stimulating the hybridomas to produce IL-2. Prior treatment of the skin with low dose UVB irradiation before epicutaneous application of contact sensitizers significantly reduced the capacity of hapten-bearing APC to stimulate the hybridomas. This observation was corroborated by results obtained from flow microfluorometry analysis of lymph node cells from FITC-sensitized mice. Lymph node dendritic cells obtained from FITC-painted mice contain a brightly staining group of cells by flow microfluorometry analysis. Lymph node dendritic cells from FITC-painted, UVB-irradiated mice did not contain this brightly staining population. These results indicate that low dose, local UVB irradiation may affect APC migration and/or function. We believe that these hybridomas will prove to be useful tools in the study of the development and regulation of contact hypersensitivity.

Studies using hapten-modified lymphoid cells as tolerogens for 1-fluoro-2,4-dinitrobenzene contact sensitization have shown that BALB/c(H-2d) mice can be made phenotypically tolerant by dinitrophenyl (DNP) on either syngeneic or allogeneic mouse lymphoid cells (DNP-LC). However, suppressor T-cell induction (Ts) in these mice (as demonstrated by adoptive transfer to syngeneic recipients) was restricted to H-2 identity between the DNP-LC and the donor mouse. It was also shown that identity at the right end of the H-2 complex was sufficient for Ts induction. In addition, this restriction was also demostrated in CBA (H-2 K) mice and for tolerance in the 1-chloro-2,4,6-trinitrobenzene contact sensitivity system using trinitrophenyl-modified lymphoid cells.

We have initiated a series of studies in vivo to investigate the physiologic role of Langerhans cells and Thy-1+ dendritic epidermal cells in the development and regulation of contact hypersensitivity. The density of I-A+ Langerhans cells and Thy-1+ dendritic epidermal cells in ammonium thiocyanate-separated epidermal sheets was determined in ten strains of inbred mice using immunofluorescence microscopy with monoclonal anti-I-A and anti-Thy-1 antibodies. Mice of different inbred strains were sensitized by painting the left ear with varying doses of either oxazolone or trinitrochlorobenzene. From five to 20 days after sensitization, groups of mice were challenged with the relevant antigen and the intensity of contact hypersensitivity, as measured by ear swelling, was determined. We then determined whether the intensity and/or duration of contact hypersensitivity in different strains of mice was influenced by the density of Thy-1+ dendritic epidermal cells or the ratio of I-A+ Langerhans cells to Thy-1+ dendritic epidermal cells. We found that there was marked variability in the density of Thy-1+ dendritic epidermal cells and I-A+ Langerhans cells in the ten strains of mice studied. The ratio of I-A+ Langerhans cells to Thy-1+ dendritic epidermal cells ranged from 0.5 in C57BL/10J mice to greater than 22 in A/J and BALB/cByJ mice. When small amounts of contact sensitizers were used to induce contact hypersensitivity (e.g., 20 micrograms of oxazolone or trinitrochlorobenzene), there was consistent strain variability in the intensity of ear swelling. There was a significant correlation between the ratio of I-A+ Langerhans cells to Thy-1+ dendritic epidermal cells and the intensity of contact hypersensitivity (Pearson's correlation coefficient 0.85, p = 0.002). Strains with the highest ratios of I-A+ Langerhans cells to Thy-1+ dendritic epidermal cells had the most ear swelling. There were no consistent differences in the duration of contact hypersensitivity observed among the ten different strains. Our results indicate that Thy-1+ dendritic epidermal cells may play a physiologic role in down-regulating contact hypersensitivity in vivo.

Immunologic suppression was induced in a mouse model of contact sensitization to DNFB by using supraoptimal doses of antigen. In these studies, in vivo measurement of ear swelling as an indication of immunologic responsiveness correlated well with measurement of in vitro antigen-induced cell proliferation. This unresponsiveness was specific, since supraoptimal doses of DNFB did not interfere with the development of contact sensitivity to another contactant, oxazolone. The decrease in responsiveness is a form of active suppression, as lymphoid cells from supraoptimally sensitized donors transferred suppression to normal recipients. Furthermore, pretreatment with cyclophosphamide (Cy) reversed the suppression seen in supraoptimally sensitized animals but had no effect on the optimal sensitization regimen. These results indicate that supraoptimal doses of contactants can activate suppressor cells and that precursors of these cells are sensitive to Cy. Such suppressors regenerate within 7 to 14 days after Cy treatment. The ability of Cy pretreatment to affect supraoptimal sensitization without affecting optimal sensitization confirms other reports indicating that the observed results of Cy treatment depend critically upon the dose of antigen used.

Genetic restrictions in generation and expression of hapten-specific suppressor cells for contact sensitivity were found. Dinitrophenol- (DNP) or trinitrophenol-modified mouse spleen cells (SC) induced suppressors in donors able to transfer suppression to normal recipients. When allogeneic DNP-SC were injected into BALB/c mice, cells were generated which were suppressive only in the allogeneic strain providing the DNP-SC. In contrast, when DNP-BALB/c-SC were injected into BALB/c mice, suppressors were generated which were active both in BALB/c and in allogeneic mice (e.g., CBA). This apparent absence of syngeneic major histocompability complex restriction may be explained by cross reactive T-cell receptors which are VH gene products.

This manuscript describes the preparation and characterization of a syngeneic antiserum raised in BALB/c mice against nylon wool-purified lymph node (LN) T cells from 2,4-dinitrofluorobenzene- (DNFB) sensitized BALB/c mice. This antiserum (anti-T DH-DNP) plus complement blocks the ability of LN or purified T cells from DNFB-sensitized donors to transfer immunity to naive recipients. The inhibitory activity of the serum is due to antibodies; these antibodies have specificity for idiotype (Id) determinants as shown by their ability to bind to purified mouse anti-DNP antibodies but not to rabbit anti-DNP or normal mouse Ig. Inhibition of transfer of immunity by the anti-Id serum is not restricted by either H-2 gene products or Igh-1 allotypes. The antiserum is antigen specific when tested on LN cells from oxazolone-sensitized mice, but has a low level of cross-reactivity against T cells from 2,4,6-trinitrochlorobenzene- (TNCB) sensitized mice. In addition, it was shown that naturally occurring anti-Id antibodies, epsilon-DNP-L-lysine, or DNP-protein (but not NIP-protein) and the anti-Id in syngeneic anti-T DH-DNP serum compete for similar binding sites on DNFB-immune T cells. We interpret these findings to indicate that immunization of mice with T cells from DNFB-sensitized syngeneic mice induces the production of anti-Id antibodies. These anti-Id antibodies are specific for determinants expressed on the T cell receptor or receptor subsite that is specific for the hapten DNP.

We investigated the mechanism(s) by which MHC-restricted suppressor T cells (Ts) induced by i.v. injection of allogeneic DNP-modified lymphoid cells (alloinduced Ts) suppress the DNFB contact sensitivity response. It was shown that alloinduced Ts acted only during the early phases (afferent limb) of sensitization. They were incapable of suppressing previously sensitized recipients or of inhibiting the expression of DNFB-immune LN cells when co-transferred into normal recipients. The target of alloinduced Ts seems to be cell proliferation, i.e., inhibition of antigen-induced cell proliferation (DNA synthesis) in Ts recipient mice. The failure of recipients of alloinduced Ts to generate DNFB-immune LN cells capable of transferring contact sensitivity to normal recipients also suggests that these Ts act by preventing the development of an expanded clone of mature immune T cells. The suppressive effects of alloinduced Ts also were inhibited by prior in vitro treatment with anti-TNP serum. The data are discussed in terms of current models of suppression, and are compared to mechanisms of suppression in other contact sensitivity models.

We investigated T-T cell interactions in the suppression of contact sensitivity. Suppressor cells that block the efferent limb of sensitivity (Ts-eff) can inhibit the passive transfer of contact sensitivity mediated by 1-fluoro-2,4-dinitrobenzene immune cells (T DH). But, Ts-eff cannot block the passive transfer of TDH which comes from cyclophosphamide (Cy) pretreated sensitized mice. We interpret these results to indicate that lymph node cells from sensitized mice contain not only TDH but also another intermediate cell which is required for the suppression of TDH by Ts-eff. This intermediate cell is sensitive to cyclophosphamide and requires antigen activation for its development. It is sensitive to adult thymectomy and anti-brain associated theta serum and is therefore designated as an auxiliary T-suppressor cell (Ts-aux). It is not sensitive to splenectomy and it carries I-J determinants. Ts-aux are required for the activity of suppressors of the efferent limb (Ts-eff) but not of suppressors of the afferent limb (Ts-aff). Thus, in the feedback loops in contact sensitivity, the generation of Tdh is coordinated with the development of auxiliary Ts which are essential for the suppression of those TDH.