Proc. Nati. Acad. Sci. USA Vol. 93, pp. 1464-1469, February 1996 Immunology Biosynthesis of major histocompatibility complex molecules and generation of T cells in Ii TAPI double-mutant mice (antigen presentation/major histocompatibility complex class I transport/major histocompatibility complex class II transport/T cell differentiation/T cell selection) SYLVIE TOURNE*t, HISSE MARTIEN VAN SANTENtt, MARIAN VAN ROON§, ANTON BERNST, CHRISTOPHE BENOIST*, DIANE MATHIS*, AND HIDDE PLOEGHi *Institut de Genetique et de Biologie Moleculaire et Cellulaire, 1 rue Laurent Fries, 67404 Illkirch, C. U. de Strasbourg, France; tMassachusetts Institute of Technology, Center for Cancer Research E17-323, 77 Massachusetts Avenue, Cambridge, MA 02139-4307; IDepartment of Anatomy and Embryology, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands; and 1Division of Molecular Genetics, Netherlands Cancer Institute, Plesmanlaan 121, 1066 XC Amsterdam, The Netherlands Communicated by Herman N. Eisen, Massachusetts Institute of Technology, Cambridge, MA, October 24, 1995 ABSTRACT Major histocompatibility complex (MHC) Class II-peptide complexes are then transported to the cell class I and II molecules are loaded with peptides in distinct surface via an as yet poorly defined route. subcellular compartments. The transporter associated with Evidence is accumulating, however, that this scenario is too antigen processing (TAP) is responsible for delivering pep- neat. Sometimes, class I molecules are capable of presenting tides derived from cytosolic proteins to the endoplasmic exogenously supplied antigens (4-7), and class II molecules reticulum, where they bind to class I molecules, while the can occasionally present endogenous antigens (8-10), al- invariant chain (Ii) directs class II molecules to endosomal though the extent to which such presentation relies on loading compartments, where they bind peptides originating mostly of class II molecules in the ER has not been accurately from exogenous sources. Mice carrying null mutations of the established. Similarly, TAP-independent presentation of ex- TAP1 or Ii genes (TAP10 or Ii°, respectively) have been useful ogenous material by class I molecules has been observed, but tools for elucidating the two MHC/peptide loading pathways. the extent to which this represents processing akin to that seen To evaluate to what extent these pathways functionally inter- for class II molecules remains to be established. It is not known sect, we have studied the biosynthesis of MHC molecules and what is responsible for such breakdown in the dichotomy, but the generation of T cells in Ii°TAP10 double-mutant mice. We some intriguing observations have been made. On the one find that the assembly and expression of class II molecules in hand, some protein epitopes are actually presented better in li° and Ii°TAP10 animals are indistinguishable and that the absence of Ii, provoking speculation that they might be formation and display of class I molecules is the same in bound to class II molecules in the ER (11). On the other hand, TAP10 and IiTAP10 animals. Thymic selection in the double Cerundolo et al. (12) and Sugita and Brenner (13) have mutants is as expected, with reduced numbers of both CD4+ detected complexes of class I molecules and Ii. These com- CD8- and CD4- CD8+ thymocyte compartments. Surpris- plexes are, in one case, diverted from the secretory pathway ingly, lymph node T-cell populations look almost normal; we and appear in the endosomes (13). propose that population expansion of peripheral T cells Here, we approach the problem by exploiting mice carrying normalizes the numbers of CD4+ and CD8+ cells in Ii°TAP1° null mutations in the TAP1 or Ii genes (TAP10 or Ti0, respec- mice. tively). In the former, class I complexes are largely held up in the ER, although a few unstable complexes, probably devoid The function of major histocompatibility complex (MHC) of peptide, appear at the cell surface; consequently, CD8+ T class I and II molecules is similar, yet distinct (for reviews see cells are poorly selected in the thymus, and there is only a refs. 1-3). Both bind antigenic peptides and present them to T paltry peripheral population (14). In the latter, class II af3 cells, but class I molecules associate primarily with peptides chains are retained in the ER, but, again, some unstable generated from cytosolic proteins and offer them to CD8+ complexes make it to the cell surface, in this case 10-30% the cytotoxic T cells, while class II molecules interact with peptides usual numbers; the CD4+ T cell compartments are reduced in derived mostly from exogenous proteins and present them to the thymus and periphery, but only by severalfold (15-17). We CD4+ helper T cells. This dichotomy reflects the intracellular have now mated Ii0 and TAP10 mice to produce litters of single pathways travelled by the two classes of MHC molecule. Class and double mutants. To explore any functional intersection of I heavy chains assemble with 132-microglobulin and are loaded the class I and II loading pathways, we compared the biosyn- with peptides shortly after being synthesized in the endoplas- thesis of MHC molecules and the generation of T cells in the mic reticulum (ER). Most of the peptides are delivered into the different littermates. ER from the cytosol by the transporter associated with antigen processing (TAP) complex. Class I-peptide complexes exit the MATERIALS AND METHODS ER and are transported along the secretory pathway, via the Golgi to the cell surface. Class II a and f3 chains assemble in Mice. Mice deficient in Ii have been described (15). The the ER and associate with the invariant chain (Ii). Ii prevents TAP10 mice used in this study were generated independently many peptides from binding in the ER and is responsible for from those previously reported (14). A 15-kb TAP1 clone was directing the exit of class II molecules from the ER and their isolated from a 129/OLA genomic phage library and analyzed targeting to endosomal compartments. In the endosomes, class by restriction mapping and partial sequencing. Two adjacent II molecules are dissociated from Ii and loaded with peptides, Bgl II restriction fragments (1400 and 700 bp, respectively), mostly processed from proteins internalized from the exterior. Abbreviations: MHC, major histocompatibility complex; TAP, trans- porter associated with antigen processing; Ii, invariant chain; ER, The publication costs of this article were defrayed in part by page charge endoplasmic reticulum; mAb, monoclonal antibody; 1D-IEF, one- payment. This article must therefore be hereby marked "advertisement" in dimensional isoelectric focusing. accordance with 18 U.S.C. §1734 solely to indicate this fact. tS.T. and H.M.v.S. contributed equally to this work. 1464 Downloaded by guest on September 26, 2021 Immunology: Tourne et al. Proc. Natl. Acad. Sci. USA 93 (1996) 1465 encoding at least the second and part of the third transmem- BrdUrd antibody (Becton-Dickinson), as described (28). All brane helix, were replaced by a 1.1-kb Bgl II-BamHI fragment other reagents are listed in refs. 27 and 28). containing the pMClneo gene (18, 19). E14 ES cells were electroporated with a 9-kbApa I targeting fragment, consisting of the neo gene flanked by 0.5 kb and 7.5 kb of TAP1 genomic RESULTS sequence at the 5' and 3' ends, respectively (Bio-Rad gene MHC Class II Molecule Assembly, Transport, and Cell pulser, 800V, 3,uF, electrode distance 0.4 cm). Transfectants Surface Expression. Assembly of class II molecules in spleno- were selected in G418, and 200 drug-resistant colonies were cytes from Ti0 mice of the H-2b haplotype is reduced, but class expanded and analyzed for homologous recombination with a II complexes can still be detected intracellularly and on the cell 750-bp HindIll-Not I fragment situated 500 bp upstream from surface (15-17). To investigate whether peptides delivered to the targeting construct. Homologous recombination had taken the ER by TAP molecules contribute to the formation and place in eight clones, and three clones were used for injection display of these class IT complexes, we performed metabolic into blastocysts. Generation of chimeras and homozygous labeling experiments and cytofluorimetric analyses on spleno- knockout mice was as described (20). cytes from Ii°/TAP10 single- and double-mutant mice com- Single and double mutants resulted from a cross between Ti° paring, in particular, Ti0 and Ii0TAP10 cells. (2 or 3 generations backcrossed onto C57Bl/6) and TAP10 A pulse-chase analysis was undertaken using a mAb that (backcrossed 2 or 3 generations onto FVB) individuals. Ani- precipitates the AabAI3b complex. As depicted in Fig. 1A, Aa mals were housed in conventional facilities in either Strasbourg and AP3 chains can be immunoprecipitated from Ii°TAP10 or Cambridge. splenocytes. Their quantity is reduced compared with wild- Metabolic Labeling and Immunoprecipitations. Freshly iso- type and TAP10 splenocytes but is approximately equal to the lated spleen cells were incubated for 45 min in methionine-free amount present in Ii0 splenocytes. The Aa and AP chains from RPMI medium, labeled for 30 min with 500 ,uCi of [35S]me- and splenocytes show a mobility shift during thionine/cysteine (1 Ci = 37 GBq) (protein labeling mix, wild-type TAP10 NEN/Du Pont), and chased in medium supplemented with 1 the chase, no doubt due to the acquisition of complex type mM unlabeled L-methionine and L-cysteine. Cells were lysed oligosaccharides in the Golgi. However, as previously reported in NP40 lysis mixture (0.5% Nonidet P-40/50 mM Tris HCl, (15-17), most of the class II complexes in Ti0 splenocytes pH 7.4/5 mM MgCl2/0.1 mM phenylmethylsulfonyl fluoride), remain in the ER, as concluded from the absence of a mobility supplemented with 10 mg of bovine serum albumin per ml. shift. Complexes in Ii°TAP10 splenocytes also appear to Lysates were precleared twice with normal mouse serum and remain largely in the ER.