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Eflicient Major Histocompatibility Complex Class I Presentation Of Proc. Natl. Acad. Sci. USA Vol. 90, pp. 4942-4946, June 1993 Immunology Eflicient major histocompatibility complex class I presentation of exogenous antigen upon phagocytosis by macrophages (vaccine/cytotoxic T lymphocyte/viral immunity/antigen presentation) MAGDALENA KOVACSOVICS-BANKOWSKI*t, KAREN CLARK*, BARUJ BENACERRAF*t, AND KENNETH L. ROCK*t *Division of Lymphocyte Biology, Dana-Farber Cancer Institute, Boston, MA 02115; and tDepartment of Pathology, Harvard Medical School, Boston, MA 02115 Contributed by Baruj Benacerraf, March 4, 1993 ABSTRACT Antigens in extracellular fluids can be pro- antigens on class I molecules (15). This activity was first cessed and presented with major histocompatibility complex defined in vitro; however, when these cells are recovered (MHC) class I molecules by a subset of antigen presenting cells from mice injected with soluble antigen, they present this (APCs). Chicken egg ovalbumin (Ova) linked to beads was antigen to class I-restricted T cells (16). The APC that presented with MHC class I molecules by these cells up to mediates this unique antigen presenting activity is a macro- 104-fold more efficiently than soluble Ova. This enhanced phage (16, 17) (unpublished data). Previous studies, reporting presentation was observed with covalently or noncovalently that the depletion of macrophages with silica or carrageenan linked Ova and with beads of different compositions. A key reduces the capacity of mice to generate a CTL response parameter in the activity of these conjugates was the size of the even to live viruses, indicate that macrophages in general beads. The APC that is responsible for this form ofpresentation may be key components in the initiation ofall CTL responses is a macrophage. These cells internalize the antigen constructs (18, 19). through phagocytosis, since cytochalasin B inhibited presen- The present studies were initiated to investigate how tation. Processing of the antigen and association with MHC antigens enter into the class I presentation pathway in these class I molecules appears to occur intracellularly as presenta- macrophages and whether this pathway could be exploited to tion was observed under conditions where there was no detect- elicit CTL immunity with nonliving antigens. able release of peptides into the extracellular fluids. When injected in vivo in C57BL/6 mice, Ova-beads, but not soluble Ova, primed CD4- CD8+ cytotoxic T lymphocytes (CTLs). MATERIALS AND METHODS Similar results were obtained in BALB/c mice immunized with Mice. Female C57BL/6 and BALB/c 5- to 8-week-old mice 13-galactosidase-beads. The implications of these findings for were purchased from The Jackson Laboratory. development of nonliving vaccines that stimulate CTL immu- Cell Lines. RF33.70 is an anti-Ova+Kb hybridoma (20). nity are discussed. EL4 is a C57BL/6 T lymphoma, and EG7 is a chicken egg ovalbumin (Ova)-transfected clone of EL4 (7). P815 is a Major histocompatibility complex (MHC) class I molecules DBA/2 mastocytoma and P13.4 is a f-galactosidase (*-Gal)- bind fragments of endogenously synthesized proteins and transfected clone of P815. EG7 and P13.4 were kindly pro- transport these peptides to the cell surface. In this manner, vided by M. Bevan (University of Washington, Seattle). cells display to the immune system a sampling of their BMA3.1A and BMC2 are C57BL/6 bone marrow macro- endogenous proteins (1-3). When a viral peptide, to which phage cell lines (unpublished data). the host is not tolerant, is presented, cytotoxic T lympho- Reagents. Ova, 3-Gal, azide, 2-deoxyglucose, and cyto- cytes (CTLs) are stimulated and kill the antigen-bearing cell. chalasin B were purchased from Sigma. Monoclonal anti- The immune system thereby purges the pathologically af- bodies (mAbs) were kindly provided by the laboratories of fected cell from the host. This CTL response plays a major origin: anti-Thy-i (M5/49) (21), anti-CD4 (GK1.5) (22), and role in host defense against pathogenic viruses and tumors (4, anti-CD8 (HO2.2ADH4) (23). 5). Antigenic Constructs. Iron oxide beads (0.5-1.5 ,um) were The peptides presented on MHC class I molecules are purchased from Advanced Magnetics (Cambridge, MA). Ova generated in a nonlysosomal compartment in cells, probably or 8-Gal was covalently linked to these beads via an amino the cytosol (3). Antigens in the extracellular fluid do not gain group bound according to the manufacturer's instructions. access into this processing compartment in most cells (6-8). These conjugates are referred to as Ova-Fe beads orj-Gal-Fe Accordingly, CTL immunity is not generally elicited when beads. Ova was linked to latex beads (Polybeads) (diameter, proteins or nonliving viruses are injected into animals (8). 0.5-10 ,um) (Polysciences) either by a covalent amino bond, This anatomical segregation of the class I pathway, while Ova-latex(C), or by passive adsorption, Ova-latex(A), ac- potentially important for preserving the immunological iden- cording to the manufacturer's directions. The amount ofOva tity of healthy cells, has been a major barrier to the devel- bound to the beads was determine based on the difference in opment ofnonliving vaccines that elicit CTL immunity. It has optical density between the solution of Ova before and after also been unclear how CD8+ immunity is elicited against the linkage, and/or by using 125I-labeled Ova, and typically certain pathogens that reside in the phagolysosomes of mac- varied from 0.1 to 9 mg/ml. rophages (9) and why sometimes exogenous antigens prime Cell Culture. APCs were exposed to antigen and T-T CTLs (10-14). hybridoma cultures were prepared essentially as described Although exogenous proteins are excluded from the class (20), except that indomethacin (0.25 ,uM) was added to the I presenting pathway in most cells, there is an antigen culture medium. Serum-free cultures were performed in presenting cell (APC) that can process and present these Opti-MEM (GIBCO) supplemented with Nutridoma (1%) The publication costs of this article were defrayed in part by page charge Abbreviations: MHC, major histocompatibility complex; CTL, cy- payment. This article must therefore be hereby marked "advertisement" totoxic T lymphocyte; APC, antigen presenting cell; Ova, chicken in accordance with 18 U.S.C. §1734 solely to indicate this fact. egg ovalbumin; 3-Gal, ,-galactosidase; mAb, monoclonal antibody. 4942 Downloaded by guest on October 1, 2021 Immunology: Kovacsovics-Bankowski et al. Proc. Natl. Acad. Sci. USA 90 (1993) 4943 (Boehringer Mannheim). In some experiments, cultures were 250 prepared in Transwell plates (Coming) with T-T hybrids and live or fixed APCs in the upper chamber separated by 0.4-,um filters from APCs and antigen in the lower chamber in serum-free medium (500 ,ul). After 24 hr, an aliquot of supernatant (100 IlI) was collected and subjected to freeze- thawing, and interleukin 2 was assayed (24). Inhibition Studies. In some experiments, macrophage cell X 10-2 10-1 100 101 102 103 10-310-210-1100 101 102 103 lines were preincubated with or without azide (30 mM) and ..150 c 250 2-deoxyglucose (5 mM) or cytochalasin B (10 ,uM) for 1 hr at C.) -D 37°C, and then antigens were added for 5 hr in the continuous ~~~~~~~200- presence of inhibitors. Subsequently, cells were fixed with 100~~~~~~~10 1% paraformaldehyde, washed with medium, and put into j/5i0 5 T-T hybridoma cultures. In Vivo Immunization. C57BL/6 or BALB/c mice were injected subcutaneously with antigen in 0.1 ml of phosphate- 0. 0 10-2 10-1 10° 101 102 103 buffered saline. The amount of antigen is indicated in the individual experimental protocols. One to two weeks later, Antigen (gg/ml) splenocytes were restimulated with EG7 (15 x 106 cells per FIG. 1. Presentation of exogenous antigens with MHC class I flask) as described (7) and splenic CTL activity was measured molecules. (A) Cultures were prepared with RF33.70 T-cell hybrid 5 days later (20). For 1-Gal, the same procedure was used (anti-Ova + Kb) (5 x 104 cells per well), thioglycolate-elicited except that P13.4 cells (6 x 106 cells per flask) were used peritoneal exudate cells (104 cells per well), and soluble Ova (open instead of EG7 cells. 51Cr release assays were performed as squares) or Ova-Fe beads (solid squares) in 96-well plates (200 PI) in described (20). duplicate. Cultures were then handled as described. (B) Cultures contained RF33.70 cells (5 x 104 cells per well), soluble Ova (open symbols), or Ova-Fe beads (solid symbols) and, as APCs (5 x 104 RESULTS cells per well), either EL4 (triangles) or BMA3.1A (squares). Cul- tures were otherwise prepared and handled as described in A. (C) Analysis of the Presentation of Exogenous Soluble Versus Cultures contained RF33.70 cells (5 x 104 cells per well), thiogly- Particulate Ova with MHC Class I Molecules. Antigens in the colate-elicited macrophages (5 x 104 cells per well), and Ova- extracellular fluids can be internalized into macrophages by latex(A). The size of the beads was 0.5 ,um (open circles), 0.75 ,um two distinct mechanisms. Soluble antigens are internalized (open triangles), 2 Am (open squares), 3 ,um (solid triangles), 6 um by endocytosis while particulate antigens can be taken up (solid squares), and 10 ,um (solid circles). Cultures were prepared and through phagocytosis. To analyze how antigens are internal- handled as described in A. (D) Cultures were prepared with BMA3.1A (5 x 104 cells per well), RF33.70 T-cell hybrid (4 x 104 ized and presented with MHC class I, we incubated APCs cells per well), and soluble Ova (open squares), 3-,m Ova-latex(A) with soluble Ova or Ova conjugated to a phagocytic sub- (open circles), or 3-,um Ova-latex(C) (solid circles).
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