Systemic treatment with anti-CD40 antibody stimulates Langerhans cell migration from the skin

Abstract

Epidermal Langerhans cells (LCs) play a pivotal role in the initiation of cutaneous immune responses. The maturation of LCs and their migration from the skin to the T cell areas of draining lymph nodes are essential for the delivery and presentation of antigen to naïve T cells. CD40, which acts as a costimulatory molecule, is present on LCs and the basal layer of keratinocytes in the skin. We show here that systemic treatment of mice with anti-CD40 antibody stimulates the migration of LCs out of the epidermis with a 70% reduction in LC numbers after 7 days, although changes in LC morphology are detectable as early as day 3. LC numbers in the epidermis returned to 90% of normal by day 21. As well as morphological changes, LC showed up-regulated levels of Class II and ICAM-1, with only minimal changes in CD86 expression 3 days following anti-CD40 treatment. Despite increased levels of Class II and ICAM-1, epidermal LC isolated from anti-CD40 treated mice were poor stimulators of a unidirectional allogeneic mixed leucocyte reaction (MLR), as were epidermal LC isolated from control mice. These results indicate that CD40 stimulation is an effective signal for LC migration, distinct from maturation of immunostimulatory function in the epidermis, which is not altered. These observations may have important implications for the mechanism of action of agonistic anti-CD40 antibodies, which have been used as an adjuvant in models of infection and experimental tumours and the primary immunodeficiency Hyper IgM syndrome caused by deficiency of CD40 ligand.

Fig. 1

The effects of anti-CD40 antibodies on epidermal Langerhans cell numbers and morphology in the ear skin of mice. (a) The numbers of LC in the epidermis at days 3, 4 and 7 days following a single i.p. injection of 250μg anti-CD40 antibody (clone 3/23) or isotype-matched control antibody (MAC193) are shown. Results are average numbers of LC ± SEM per mm² measured in 10 digital images taken at random from 2 ears per group and are from one representative experiment of at least three performed. □ MAC 193; ; anti-CD40. P-values are: *P = 1·6 × 10⁻¹¹, **P = 2·3 × 10⁻¹⁵. (b) The numbers of LC in the epidermis at days 1, 3, and 7 following a single i.p. injection of 250 μg moAb 1C10 (anti-CD40) are shown. The control moAb, MAC 193, had no effect on LC numbers and the data on days 1, 3 and 7 have been pooled. P-values are: *P = 4 × 10⁻⁸, **P = 1·1 × 10⁻²⁵. Results are as described in legend to Fig. 1a. (c) Langerhans cells stained with MHC Class II FITC in epidermal sheets of anti-CD40 (3/23) and control moAb (MAC193)-treated mice at days 3, 4 and 7 after a single i.p. injection of 250 μg antibody (original magnification × 200). Increases in the size of LC and MHC Class II expression are seen on days 3 and 4 before effects on LC numbers, which are most marked by day 7. (d) CD40 knockout and 129/Sv wildtype mice were given a single i.p. injection of anti-CD40 antibody (3/23) or control moAb (MAC193) and the numbers of LC counted on day 7. The statistical difference in the LC numbers between the anti-CD40 treated control and CD40 knockout mice is *P = 3·1 × 10⁻⁵. Results are as described in legend to Fig. 1a.

Fig. 1

The effects of anti-CD40 antibodies on epidermal Langerhans cell numbers and morphology in the ear skin of mice. (a) The numbers of LC in the epidermis at days 3, 4 and 7 days following a single i.p. injection of 250μg anti-CD40 antibody (clone 3/23) or isotype-matched control antibody (MAC193) are shown. Results are average numbers of LC ± SEM per mm² measured in 10 digital images taken at random from 2 ears per group and are from one representative experiment of at least three performed. □ MAC 193; ; anti-CD40. P-values are: *P = 1·6 × 10⁻¹¹, **P = 2·3 × 10⁻¹⁵. (b) The numbers of LC in the epidermis at days 1, 3, and 7 following a single i.p. injection of 250 μg moAb 1C10 (anti-CD40) are shown. The control moAb, MAC 193, had no effect on LC numbers and the data on days 1, 3 and 7 have been pooled. P-values are: *P = 4 × 10⁻⁸, **P = 1·1 × 10⁻²⁵. Results are as described in legend to Fig. 1a. (c) Langerhans cells stained with MHC Class II FITC in epidermal sheets of anti-CD40 (3/23) and control moAb (MAC193)-treated mice at days 3, 4 and 7 after a single i.p. injection of 250 μg antibody (original magnification × 200). Increases in the size of LC and MHC Class II expression are seen on days 3 and 4 before effects on LC numbers, which are most marked by day 7. (d) CD40 knockout and 129/Sv wildtype mice were given a single i.p. injection of anti-CD40 antibody (3/23) or control moAb (MAC193) and the numbers of LC counted on day 7. The statistical difference in the LC numbers between the anti-CD40 treated control and CD40 knockout mice is *P = 3·1 × 10⁻⁵. Results are as described in legend to Fig. 1a.

Fig. 2

The effects of anti-CD40 antibodies on the phenotype of epidermal Langerhans cells. (a) BALB/c mice injected with either anti CD40 antibody (3/23) or control antibody (MAC 193) were killed after 3 days. Epidermal cell suspensions were made, the cells stained for MHC Class II, CD86 and ICAM-1 and analysed by flow cytometry. The expression of MHC Class II, CD86 and ICAM-1 on fresh Langerhans cells isolated from ear skin of normal mice were compared. The level of expression of these markers of maturity is increased in the CD40 treated LC. (b) In vitro matured epidermal Langerhans cells isolated from 48h skin explant organ cultures and migrated LC were used to compare the expression of CD86. The expression of CD86 is increased in the CD40 treated LC but not to the extent of the epidermal LC which have a bimodal expression of CD86 or the migrated LC derived from explants.

Fig. 3

The effects of anti-CD40 antibodies on lymph node dendritic cell numbers. BALB/c mice injected with either (a) anti-CD40 antibody (3/23) or (b) control antibody (MAC 193) were killed on day 7. Lymph nodes were removed and cell suspensions were enriched for dendritic cells by metrizamide gradient centrifugation. Dendritic cells were identified by coexpression of MHC Class II and NLDC-145. The double positive cells in the control antibody treated group is 7% compared with 22% in the CD40 treated mice.

Fig. 4

The effects of anti-CD40 antibodies on antigen presenting cell function of purified epidermal Langerhans cells. (a) BALB/c mice injected with either anti CD40 antibody (3/23) or control antibody (MAC 193), were killed on day 3. Epidermal cell suspensions were stained with anti-CD32 FITC and anti-MHC Class II PE to verify the anti-CD32 staining of LC. (b) Epidermal cell suspensions were made and the cells stained with CD32 FITC and sorted to >90% purity by flow cytometry. (c) Sorted LC were used as stimulators in a unidirectional MLR. In vitro matured and sorted LC which had migrated from 48 h skin explant organ cultures were used as a positive control (these had also been subjected to trypsin). ○ Control Ab; • Anti-CD40; ▪ Migrated cells Results are mean proliferation index of each triplicate ± SEM from a representative experiment which has been repeated 3 times with similar results.