Cutting edge: MHC class II expression by pulmonary nonhematopoietic cells plays a critical role in controlling local inflammatory responses

Abstract

The interaction of CD4(+) T cells with MHC class II (MHCII)-expressing hematopoietic APCs plays a critical role in both the generation of protective immune responses and maintenance of tolerance in the lung. The functional significance of MHCII expression by nonhematopoietic stromal cells, however, has not been defined in vivo. Using a novel mouse model of orthotopic left lung transplantation, we demonstrate that selective elimination of MHCII expression on nonhematopoietic cells leads to an inflammatory response as a result of reduced peripheral generation of regulatory CD4(+) T cells. Absence of MHCII expression on nonhematopoietic cells also inhibits local growth of metastatic pulmonary tumor. These findings indicate that nonhematopoietic cells play a previously unrecognized role in downregulating inflammatory responses in nonlymphoid tissues.

Figure 1

MHC Class II expression in the lung. (A) MHCII on pulmonary CD45⁻ non-hematopoietic cells. (B) Immunostaining for CD31 (FITC) and MHC II (Texas Red) in lungs of B6 chimeras after reconstitution with B6II⁻ hematopoietic cells. (C) CD45 expression on MHCII-positive cells in lungs and mediastinal lymph nodes. (D) Invariant chain expression by pulmonary non-hematopoietic cells (black line-antibody; shaded grey isotype). (E) DQ-ovalbumin processing and cleavage as identified by green fluorescence in live CD45⁻MHCII⁺ cells (thick green line) compared to cells fixed in 5% paraformaldehyde prior to incubation (dotted green line) or unlabeled cells (shaded grey plot). (F) Substitution of donor with recipient-derived hematopoietic APCs and CD4⁺ T cells in left lung grafts. (G) MHC class II expression on non-hematopoietic cells in transplanted B6 and B6II⁻ lungs. Analysis is representative of at least four separate experiments.

Figure 2

Analysis of B6 and B6II⁻ lung grafts. (A) Hematoxylin and eosin staining of perivascular and peribronchial infiltrates (arrows) in a B6II⁻ or B6 graft (100x). (B) Absolute T cell numbers (top) and percentage of effector memory T cells (bottom) in grafts (B6=blue; B6II⁻=red). (C) Percentage of congenic CD62L^loCD44^hi CD4⁺ effector memory T cells generated from adoptively transferred CD62L^hiCD44^lo CD4⁺ naïve T cells in lung grafts and mediastinal lymph nodes of B6 and B6II⁻ recipients. (D) Hematoxylin and eosin staining (100x) staining of B6 or B6II⁻ lungs grafted into B6/B6II⁻ F1 recipients with arrow depicting perivascular cuffing in B6II-graft. (E) Total number of CD4⁺ T cells and percent CD4⁺CD62L^loCD44^hi effector memory T cells in B6 and B6II⁻ grafts transplanted into B6/B6II⁻ F1 recipients. (F) Absolute CD8⁺ T cell numbers (top) and percentage of CD62L^loCD44^hi CD8⁺ T cells (bottom) in lung grafts of CD4⁺ T cell-depleted B6 recipients. (G) Percentages of CD4⁺ T cells expressing Foxp3 and CD8⁺: CD4⁺ Foxp3⁺ T cell ratios in B6 and B6II⁻ grafts. (H) Percentages of CD4⁺ T cells expressing Foxp3 (top) and perivascular and peribronchial infiltrate (arrows) of B6 grafts two weeks after transplantation into hosts treated with 1mg PC61 i.p day −2, and 250 μg day −1 and every three days thereafter (bottom) (100x). (I) Representative plots demonstrating adoptively transferred cells with de novo expression of Foxp3. (J) Graphic representation of CD4^+Foxp3GFP− cells converting to CD4^+Foxp3GFP+ cells in B6^45.1 (blue) and B6II^−45.1 (red) grafts (top) and similar data demonstrating de novo Foxp3 expression in adoptively transferred OT-II CD4^+Foxp3− T cells after instillation of saline (left) or 1 mg of ovalbumin (right) intratracheally into B6^45.1 recipients of lung grafts (bottom). Analyses representative of at least eight mice per group except where indicated in the graph.

Figure 3

Tumor immune response. Gross appearance and weights of B6 and B6II⁻ grafts and native right lungs (A) without and (B) after injection of B16 melanoma. Hematoxylin and eosin staining of grafts indicating melanoma (labeled as tumor) and inflammatory infiltrates (arrows). (C) Gross appearance and weights of B6 and B6II⁻ grafts four weeks after transplantation into B6 Rag^−/− mice injected with B16 melanoma. (D) Absolute numbers of graft-infiltrating CD4⁺CD90⁺ T cells and percentage of graft-infiltrating CD4⁺ T cells expressing Foxp3 in B6 or B6II⁻ grafts at same time points as shown in (B). (E) Absolute numbers of CD8⁺CD90⁺ T cells and CD8⁺ T cells with T cell receptor-specificity for tyrosinase-related protein 2 in B6 or B6II-grafts at same time points as described in (B). Figure summarizes sixteen and nineteen transplanted mice per group in B6 recipients of B6 and B6II⁻ grafts, respectively and seven transplanted mice per group in B6 Rag^−/− recipients.