Immunotherapy with dendritic cells directed against tumor antigens shared with normal host cells results in severe autoimmune disease

Abstract

Vaccination with dendritic cells (DCs) presenting tumor antigens induces primary immune response or amplifies existing cytotoxic antitumor T cell responses. This study documents that antitumor treatment with DCs may cause severe autoimmune disease when the tumor antigens are not tumor-specific but are also expressed in peripheral nonlymphoid organs. Growing tumors with such shared tumor antigens that were, at least initially, strictly located outside of secondary lymphoid organs were successfully controlled by specific DC vaccination. However, antitumor treatment was accompanied by fatal autoimmune disease, i.e., autoimmune diabetes in transgenic mice expressing the tumor antigen also in pancreatic beta islet cells or by severe arteritis, myocarditis, and eventually dilated cardiomyopathy when arterial smooth muscle cells and cardiomyocytes expressed the transgenic tumor antigen. These results reveal the delicate balance between tumor immunity and autoimmunity and therefore point out important limitations for the use of not strictly tumor-specific antigens in antitumor vaccination with DCs.

Figure 1

DC treatment of peripherally growing MC-GP tumors leads to diabetes in RIP-GP mice. Mice with established MC-GP tumors (diameter >5 mm on day 14) were used for immunotherapy. Closed symbols indicate blood glucose levels; open symbols represent tumor volume in three individual mice at the indicated time points after beginning of treatment with H8-DC. (A) Tumor growth and blood glucose levels in untreated RIP-GP mice. (B) Successful treatment of MC-GP tumors with GP33-expressing H8-DCs in RIP-GP mice is accompanied by rapidly developing hyperglycemia. Mice were immunized intravenously with 2 × 10⁵ H8-DCs on days 0, 2, 10, and 12. (C) Development of hyperglycemia in RIP-GP mice after repetitive intravenous immunization with 2 × 10⁵ H8-DCs on days 0, 2, 10, and 12. Results from one of three comparable experiments are shown.

Figure 2

Immune responses against the β-galactosidase antigen in SM-LacZ mice. (A) SM-LacZ mice or control C57BL/6 mice (B6) were intravenously immunized with 2 × 10⁶ pfu of VV-LacZ. 8 d later, spleen cells were restimulated in vitro for 5 d with peptide-labeled, irradiated spleen cells. Specific lysis was measured on β-gal 497–504-labeled EL4 target cells (closed symbols) or on EL4 cells without peptide (open symbols). Data from two individual mice per group are shown. (B) Rejection of β-gal 497–504-pulsed spleen cells. Naive or primed SM-LacZ mice or C57BL/6 control mice (intravenous injection of 2 × 10⁵ β-gal 497–504-pulsed DCs) were intravenously transfused with 4 × 10⁷ β-gal 497–504-pulsed spleen cells (β-gal⁺) labeled with a high fluorescence intensity and with 4 × 10⁷ control spleen cells labeled with a low fluorescence intensity (ctrl). 24 h after adoptive transfer, PBLs were analyzed by FACS^® analysis. Results are from one of two comparable experiments.

Figure 3

Characterization of EL4-LacZ tumor cells and immunotherapy of EL4-LacZ tumors in SM-LacZ mice. (A) EL4-LacZ (•) and EL4 cells with (▪) or without (□) peptide β-gal 497–504 were used in vitro as targets in a ⁵¹Cr-release assay. Effector cells were β-gal 497–504-restimulated spleen cells from C57BL/6 mice infected with 2 × 10⁶ pfu VV-LacZ. (B) 2 × 10⁷ EL4-LacZ tumor cells were injected subcutaneously into the flanks of C57BL/6 mice. 8 d later, splenocytes were restimulated in vitro using irradiated, β-gal 497–504-pulsed spleen cells for 5 d, and the CTL activity was determined in a ⁵¹Cr-release assay on β-gal 497–504-labeled EL4 target cells (closed symbols) or on EL4 cells without peptide (open symbols). (C) Small pieces of EL4-LacZ tumors were implanted subcutaneously in the flanks of SM-LacZ mice on day 1, and tumor growth was assessed at the indicated time points. (D) SM-LacZ mice received small EL4-LacZ tumor pieces as in C and were treated from day 0 repetitively by intravenous injection with 3–5 × 10⁵ β-gal 497–504-pulsed DCs. Arrows, day of injection. Numbers indicate the proportion of tumor rejection (or tumor growth) in tumors tested. Results from one of two comparable experiments are shown.

Figure 4

Immunohistological analysis of heart and lung tissue in SM-LacZ mice transplanted with EL4-LacZ tumors and treated repetitively with 3–5 × 10⁵ β-gal 497–504-pulsed DCs (A–F, J, K) or left untreated (G–I). Formalin-fixed and paraffin-embedded sections of heart (A) and lung (B) tissue were stained for smooth muscle actin from a DC-treated SM-LacZ mouse on day 11 after immunization. The inset in A is a magnification of the boxed area in A showing a strong lymphocytic infiltrate between heart muscle cells (arrows) and in the interstitium surrounding coronary arteries (arrowhead). The inset in B is a magnification of the boxed area in B stained for factor VIII showing the strongly increased adhesion of lymphocytes to the inflamed endothelium (arrows) and a strong infiltrate of lymphocytes in the arterial adventitia. Staining for CD8⁺ lymphocytes in the heart (C), lung (D), and tumor (E and F) of a DC-treated SM-LacZ mouse on day 11. (E) Strong CTL infiltration in a regressing tumor and (F) infiltration of CTL in a growing tumor. Arrows in F indicate tumor infiltrating lymphocytes; the arrowhead indicates an artery not infiltrated by CD8⁺ lymphocytes. In untreated SM-LacZ mice with transplanted EL4-LacZ tumors, CD8⁺ lymphocytes were not detected in the right ventricle wall (G) or pulmonary arteries (H) but were occasionally found in small numbers in the tumor tissue (I, arrow). (J) Dilated right ventricle in SM-LacZ mouse 25 d after repetitive immunization with β-gal 497–504-pulsed DCs. Inset is a magnification of the boxed area in J showing infiltration with mononuclear cells, fibrotic tissue, and remaining intact cardiomyocytes. (K) Macroscopic pathology of two hearts from DC-treated SM-LacZ mice on day 25 (top row) in comparison with two control hearts from untreated animals without tumors (bottom row). Ruler indicates the size in millimeters. Original magnifications: A and B, ×50; J, ×20; insets in A, B, and J, ×200; C–I, ×100.