Langerhans cells are not required for graft-versus-host disease

Abstract

Graft-versus-host disease (GVHD) is initiated and maintained by antigen-presenting cells (APCs) that prime alloreactive donor T cells. APCs are therefore attractive targets for GVHD prevention and treatment. APCs are diverse in phenotype and function, making understanding how APC subsets contribute to GVHD necessary for the development of APC-targeted therapies. Langerhans cells (LCs) have been shown to be sufficient to initiate skin GVHD in a major histocompatibility complex-mismatched model; however, their role when other host APC subsets are intact is unknown. To address this question, we used mice genetically engineered to be deficient in LCs by virtue of expression of diphtheria toxin A under the control of a BAC (bacterial artificial chromosome) transgenic hu-man Langerin locus. Neither CD8- nor CD4-mediated GVHD was diminished in recipients lacking LCs. Similarly, CD8- and CD4-mediated GVHD, including that in the skin, was unaffected if bone marrow came from donors that could not generate LCs, even though donor LCs engrafted in control mice. Engraftment of donor LCs after irradiation in wild-type hosts required donor T cells, with immunofluorescence revealing patches of donor and residual host LCs. Surprisingly, donor LC engraftment in Langerin-diphtheria toxin A (DTA) transgenic hosts was independent of donor T cells, suggesting that a Langerin(+) cell regulates repopulation of the LC compartment.

Figure 1

Recipient Langerhans cells are not required for clinical or histologic GVHD in the C3H.SW→B6 model. Tg⁺ and Tg⁻ hosts were irradiated and reconstituted with C3H.SW BM with or without 2-3 × 10⁶ purified C3H.SW CD8 cells. Shown are data from 1 of 2 experiments with comparable results. Tg⁺ and Tg⁻ CD8-recipients had similar weight change (A; P > .170 comparing Tg⁺ and Tg⁻ CD8 recipients at all time points; P < .01 comparing each CD8 group to its respective BM alone control from day +20 onward), incidence of clinical skin disease (B; P = .299 comparing Tg⁺ and Tg⁻ CD8 recipients; P < .001 comparing each CD8 recipient to its BM alone control), and number of skin ulcers (C). (D) GVHD pathology scores in skin, ear, liver, and colon. The P values for number of skin ulcers and pathology scores are shown in the figure.

Figure 2

Recipient Langerhans cells are not required for clinical or histologic GVHD in the B6^bm12→B6 model. Tg⁺ and Tg⁻ hosts were irradiated and reconstituted with B6^bm12 BM with or without B6^bm12 splenocytes containing 10⁶ CD4 cells. (A) Percentage weight change in 2 independent experiments. P > .07 comparing Tg⁺ and Tg⁻ CD4 recipients at all time points; P < .05 comparing each CD4 group to its respective BM-alone control from day +13 onward. (B) Pathology scores from the 2 experiments combined; P values are shown in the figure. Serum samples collected from predesignated mice on day +7 after BMT were analyzed for cytokine levels (C; 3 samples per group; *P = .05 comparing BM-alone to CD4 recipients; P = .7 comparing CD4-recipient groups; data from 1 of 2 experiments with similar results).

Figure 3

Donor T cells induce variable turnover of Langerhans cells. Ears from transplanted mice were analyzed for LC turnover by the LC-migration assay (A-B) and by immunofluorescence (C). To identify LCs (A), we gated on ethidium monoazide-negative cells (left panel) that were Langerin⁺MHCII⁺ (second panel) Note that nearly all MHCII⁺ cells express Langerin. We gated on MHCII⁺Langerin⁺ cells and determined the fractions that were host- or donor-derived based on expression of CD45.1 or CD45.2 (right panels). (B) Scatter plot of data analyzed as per panel A; each symbol represents data from an individual mouse. P < .03 comparing T-cell recipients to their respective BM-alone controls (data combined from 2 independent experiments with similar results). (C) Immunofluorescence staining. Epidermal preps were costained for expression of CD45.2 (red), MHCII (green), and Langerin (gray). Langerhans cells are present in recipients of BM only, but they are host-derived (CD45.2⁻; top row). With donor CD8 cells, donor CD45.2⁺ LCs engraft (second row), but engraftment is focal. Shown is an area of donor LCs abutting residual host LCs (see overlay of CD45.2 and MHCII staining in the third panel). The bottom 2 rows are staining of samples from CD45.1 and CD45.2 control mice. Images were originally captured with 200× magnification.

Figure 4

Donor Langerhans cells engraft in Langerin-DTA mice independent of donor T cells. (A-B) Flow cytometric analysis of LCs. LCs are present in ears of Langerin-DTA mice transplanted with T cell–depleted C3H.SW BM (A) (representative flow cytometry). Donor LCs engraft in Langerin-DTA recipients of Tg⁻ BM (B third panel) or RAG1^−/− BM (B fourth panel). The first 2 panels show data from unmanipulated B6 and Tg⁺ control mice. Data are representative of 3-6 mice per group from at least 2 independent experiments with similar results. (C) Epidermal preparations from B6 controls or RAG1^−/−→Tg⁺ transplantation recipients were stained for Langerin (red, first column) and MHCII (green, second column); the overlay of Langerin and MHCII expression is shown in the third column. Note the patchy engraftment of donor LCs in RAG1^−/−→Tg⁺ recipients.

Figure 5

Donor Langerhans cells are not required for GVHD in the C3H.SW→B6 strain pairing. B6 mice were irradiated and reconstituted with Tg⁺ or Tg⁻ C3H.SW BM with or without Tg⁺ C3H.SW CD8⁺ T cells. Clinical GVHD was similar in both CD8-recipient groups as measured by percentage weight loss (A; P > .146 comparing CD8 recipients of Tg⁺ or Tg⁻ BM from day 14 onward; P < .021 comparing each CD8 group to its respective BM alone control from day 22 onward), incidence of skin disease (B; P = .514 comparing CD8 recipients of Tg⁺ and Tg⁻ BM; P < .004 comparing each CD8 recipient to its BM-alone control) and the number of skin ulcers (C; P = .234 comparing CD8 recipients of Tg⁺ and Tg⁻ BM; P < .04 comparing each CD8 recipient to its BM-alone control). (D) Histopathology scores. P values are noted in the figure.

Figure 6

Similar GVHD in C3H.SW Tg⁻→B6 Tg⁻ and C3H.SW Tg⁺→B6 Tg⁺ CD8 recipients. GVHD in Tg⁻→Tg⁻ and Tg⁺→Tg⁺ transplantations was similar as measured by percentage weight change (A; P > .13 comparing Tg⁺ and Tg⁻ CD8 recipients from day 8 onward; P < .02 comparing each CD8 group to its respective BM-alone control from day 26 onward), incidence of skin disease (B; P = .41 comparing Tg⁺ and Tg⁻ CD8 recipients; P < .014 comparing each CD8 recipient to its BM-alone control), and number of skin ulcers (C; P = .91 comparing Tg⁺ and Tg⁻ CD8 recipients; P < .036 comparing each CD8 recipient to its BM-alone control). (D) Pathology scores. P values are noted in the figure.

Figure 7

Donor Langerhans cells are not required for GVHD in the B6.C→BALB/c strain pairing. BALB/c mice were irradiated and reconstituted with Tg⁺ or Tg⁻ B6.C BM with or without Tg⁺ B6.C CD4 cells. (A) Percentage weight loss. Two independent experiments are shown as left and right panels. Data were not combined due to differences in the kinetics and magnitude of weight loss in the 2 experiments. Experiment 1: P < .028 comparing each CD4 recipient group to its respective BM-alone group at all measurements; P > .05 comparing Tg⁺ and Tg⁻ CD4 recipients at all measurements except days 13, 15, and 34). Experiment 2: P < .026 comparing each CD4-recipient group to its respective BM-alone group from day +33; P > .05 comparing percentage weight loss of Tg⁺ and Tg⁻ CD4 on all days except day +2 and days +16 to +23. (B-C) Combined data from 2 experiments shown in A. (B) Percentage unaffected by skin disease (P < .003 comparing each CD4 recipient group to its respective BM-alone groups; P > .384 comparing Tg⁺ and Tg⁻ CD4-recipient groups). (C) Clinical scores of mice affected by skin GVHD (P > .179 comparing Tg⁺ and Tg⁻ CD4 recipient groups at all time points). (D) Pathology scores. P values are noted in the figure.