Donor lung derived myeloid and plasmacytoid dendritic cells differentially regulate T cell proliferation and cytokine production

Abstract

Background: Direct allorecognition, i.e., donor lung-derived dendritic cells (DCs) stimulating recipient-derived T lymphocytes, is believed to be the key mechanism of lung allograft rejection. Myeloid (cDCs) and plasmacytoid (pDCs) are believed to have differential effects on T cell activation. However, the roles of each DC type on T cell activation and rejection pathology post lung transplantation are unknown.

Methods: Using transgenic mice and antibody depletion techniques, either or both cell types were depleted in lungs of donor BALB/c mice (H-2(d)) prior to transplanting into C57BL/6 mice (H-2(b)), followed by an assessment of rejection pathology, and pDC or cDC-induced proliferation and cytokine production in C57BL/6-derived mediastinal lymph node T cells (CD3+).

Results: Depleting either DC type had modest effect on rejection pathology and T cell proliferation. In contrast, T cells from mice that received grafts depleted of both DCs did not proliferate and this was associated with significantly reduced acute rejection scores compared to all other groups. cDCs were potent inducers of IFNγ, whereas both cDCs and pDCs induced IL-10. Both cell types had variable effects on IL-17A production.

Conclusion: Collectively, the data show that direct allorecognition by donor lung pDCs and cDCs have differential effects on T cell proliferation and cytokine production. Depletion of both donor lung cDC and pDC could prevent the severity of acute rejection episodes.

Figure 1

cDC and/or pDC depletion setup in murine orthotopic transplant model. Donor →recipient transplanted groups: Wt BALB/c→Wt C57BL/6; DT (50 ng) injected DTR-Tg BALB/c→Wt C57BL/6; anti-mPDCA-1 Ab (500 ng) injected Wt BALB/c→Wt C57BL/6; anti-mPDCA-1 Ab (500 ug) and DT (50 ug) injected DTR-Tg BALB/cc→Wt C57BL/6. N = four to six mice per group.

Figure 2

Verification of cDC or pDC depletion by flow cytometry. Lung mononuclear cells were stained for DC subset markers. Cells are reported as percent of total population. Macrophages were excluded using FL1 autofluorescence and the macrophage specific marker F4/80. A) Myeloid dendritic cells: B220^-, Gr1^-, CD11b⁺ and CD11c⁺. Wt BALB/c + DT (non-depleted control) vs. DTR Tg BALB/c + DT (depleted) yield a 63% depletion. B) Plasmacytoid dendritic cells: CD11clow, B220⁺, and PDCA⁺. Wt + isotype Ab (non-depleted control) vs. Wt Balb/c + mPDCA-1 Ab (depleted) yields a 84% depletion. Data are representative of four to six mice in each group.

Figure 3

T cell alloreactivity in response to cDC or pDC stimulation. Lung transplantation was performed as described in Methods and transplanted lungs harvested at 7 days post transplant. A-D) Mediastinal lymph node CD3+ T cells from lung transplant recipient (C57BL/6) mice were cultured alone (3 × 10⁵) or in the presence of Wt donor (BALB/c)-derived cDCs or pDCs (3 × 10⁴). Cells were incubated for 72 h at 37°C. T-cell proliferation was measured by 3H thymidine incorporation. A) Spontaneous T cell proliferation of Wt, DTR, PDCA1 and DTR/pDCA1 transplant groups. B) cDC and pDC were co-cultured with Wt T cells isolated from lung transplant recipients that received dendritic cell sufficient lung allografts. C) cDC and pDC co-cultured with recipient-derived T cells isolated from cDC, pDC or cDC/pDC depleted transplant groups. D) pDC and cDC co-cultured with recipient-derived T cells isolated from cDC and pDC depleted transplant groups. Responses of recipient cells were compared using one-way ANOVA analysis. ** p < 0.01, # p = 0.01, ## p = 0.02 * p < 0.05 Panels E-H represent H&E stained lung sections from allografts harvested at day 7. Table 2) Histologic rejection scores for transplant groups. Data represent the mean +/- SD of "A" scores for four to six mice in each group harvested at 7 days. *p < 0.05 compared to other groups. (40× magnification).

Figure 4

Cytokine production in response to cDC or pDC stimulation. Mediastinal lymph node CD3⁺ T cells from lung transplant recipient (C57BL/6) mice were cultured alone (3 × 10⁵) or in the presence of Wt donor (BALB/c)-derived cDCs or pDCs (3 × 10⁴). Cells were incubated for 72 h at 37°C. Culture supernatant was collected and A) IFN-γ, B) IL-6, C) IL-10, and D) IL-17A were measured by cytometric bead assay. * p < 0.01, # p < 0.05 compared to cDC + Wt T and pDC + Wt T groups by one-way ANOVA with Dunnett's Multiple Comparison post-test.