Hans-Georg Rammensee

MHC ligands and peptide motifs

1. History and Overview -- 2. The MHC Genes -- 3. The Structure -- 4. The Function -- 5. Recognition by Immune Cells -- Appendix A: Useful Internet Addresses -- Appendix B: Computer Programs -- Appendix C: Abbreviations.

Pool sequencing of natural HLA-DR, DQ, and DP ligands reveals detailed peptide motifs, constraints of processing, and general rules.

We have approached the problem of MHC class II ligand motifs by pool sequencing natural peptides eluted from HLA-DR, DQ, and DP molecules. The results indicate surprisingly clear patterns, although not quite as clear as with natural class I ligands. The most striking feature is a highly dominant Proline at position 2. We interpret this to be a consequence of aminopeptidase N-like activity in processing. Another general aspect is the existence of three to four hydrophobic or aromatic anchors, whereby the first and the last are separated by five to eight residues. The peptide motifs for HLA-DR1, DR5, DQ7, and DPw4 are allele-specific and differ by spacing and occupancy of anchors. The anchors tend to be flanked by clusters of charged residues, and small residues, especially Ala, are frequent in the motif centers. These detailed motifs allow one to interpret most previous (DR-) motifs as fitting one or more of the anchors or conserved clusters. The relative motif symmetry suggests the possibility of bidirectional binding of peptides in the class II groove.

MHC molecules as peptide receptors

The central unit for regulation of the specific immune system is a trimolecular complex made up of the T cell antigen receptor, the MHC molecule, and the MHC ligand. The third component is a peptide derived as a degradation product from a protein. During recent years there has been some progress in understanding the interaction between MHC molecules and their peptide ligands: MHC molecules are peptide receptors of peculiar specificity, being able to accommodate millions of different peptides provided they share some common features.

Dominant aromatic/aliphatic C-terminal anchor in HLA-B*2702 and B*2705 peptide motifs

The peptide motifs of HLA-B*2702 and B2705 molecules were determined by pool sequencing of natural ligand mixtures, using established methods (Fa!k et al. 1991). B2702 molecules were immunoprecipitated from C 1R cells transfected with the B2702 gene (a gift from P. Cresswell), using either W6/32 antibodies (specific for HLA-A, B, C; Barnstable et al. 1978) or TM5 antibodies, specific for HLA-B27 (Thurau et al. 1989). As a source of B2705 molecules, the following cells were used: LG2, expressing HLA-B*2705 (Jardetzky et al. 1991); C1R cells transfected with B2705 (a gift from P. Cresswell); or a mouse (BALB/c)-derived Bcell lymphoma, X63, transfected with the B2705 gene (a gift from E. Weiss). Antibodies used for precipitation of B* 2705 molecules were PA2.6 (Parham and B odmer 1978), or W6/32 (both recognizing HLA-A, B, C), or TM5. In some instances, LG2 lysates were precleared with BB7.2 antibodies (Parham and Brodsky 1981) to remove A2 molecules also expressed by LG2 cells. Ligands were extracted with trifluoroacetic acid; ligand separation was done either on a 4.0 mm or on a 2.1 mm high pressure liquid chromatography reversed phase column using Pharmacia (Freiburg, Germany) equipment; the ligand mixture was sequenced on a model 477/120 (Applied Biosystems, Weiterstadt, Germany), all as described (Falk et al. 1991). Individual ligands were sequenced on a model 476 sequencer (Applied Biosystems).

Peptide motifs of HLA-B35 and-B37 molecules

MHC molecules are peptide receptors of peculiar specificity (Falk et al. 1991; for review, see Rammensee et al. 1993). Each MHC allelic product has its individual peptide specificity, summarized as a peptide motif that is characterized by the position and occupancy of anchor residues whose side chains protrude into complementary pockets inside the peptide-accomodating groove of the respective MHC molecule. Many class I molecules, in addition, show a preferential peptide length. The most convenient way to obtain information on the peptide motif of a given MHC molecule is to elute its natural ligands, and to sequence them as a pool (Falk et al. 1991). Conserved positions, or anchor positions, will stand out as dominant sequencing signals. A more laborious way is to isolate and to sequence a number of individual peptide ligands, to align their sequences, and to analyze whether any position is outstanding by virtue of its being frequently occupied by the same or similar residues (Jardetzky et al. 1991). Here we describe the peptide motifs of HLA-B35 and -B 37, both obtained by pool sequencing (Falk et al. 1991). The B3501 as well as the B3701 gene were transfected into mouse P815 cells (Nelson et al. 1991; D. Schendel, unpublished observation). Transfectants were grown to high numbers in roller bottle tissue culture and the human class I molecules were precipitated from detergent lysates of the cells, using PA2.6 antibodies specific for HLA-A, -B, and -C (Parham and Bodmer 1978). Peptides were dissociated from the precipitate by treatment with TFA, and the peptide-containing supernatant was separated by reversed phase high pressure liquid chromotography (HPLC; Falk et al. 1991). The fractions known from previous experience to contain natural MHC ligands were pooled and

Uneven tissue distribution of minor histocompatibility proteins versus peptides is caused by MHC expression.

Naturally processed minor histocompatibility (H) peptides corresponding to H-4b, H-Y, and an unmapped BALB.B minor H gene were quantified in a relative way in 15 different tissues of male BALB.B mice. For one of these minor H antigens, we could also determine the relative content of the respective protein. For each minor H peptide, an individual tissue distribution was found. Tissues expressing little or no MHC (major histocompatibility complex), like brain, contained only small amounts of minor H peptides or none at all, although the same tissues contained minor H protein in substantial quantities. By contrast, Kb-expressing brains from mice transgenic for Kb under control of the glial acidic protein promoter contained both minor H peptide and protein in high amounts. Thus, the expression of minor H peptides in a given tissue is dependent on coexpression of the restricting MHC class I molecules.

Protein-specific cytotoxic T lymphocytes. Recognition of transfectants expressing intracellular, membrane-associated or secreted forms of β-galactosidase

BALB/c-derived tumor cells were transfected with recombinantEscherichia coli β-galactosidase (β-gal) genes which were inserted into IgM heavy chain gene derivatives, leading to expression of the resulting fusion protein in different cellular compartments. A β-gal-specific, major histocompatibility complex (MHC) class I-restricted CD8+CD4− cytotoxic T lymphocyte (CTL) line of BALB/c origin raised against one transfectant expressing cytoplasmic β-gal also lysed transfectants expressing β-gal as membrane-inserted fusion protein, as well as transfectants secreting β-gal. Our data show that MHC class I-restricted CTL can recognize fragments of nonviral cellular proteins, be they expressed as intracellular, membrane-inserted, or secreted products. The findings confirm and extend a hypothesis on the nature of minor histocompatibility (H) antigens formulated earlier.

The making of the dominant MHC class I ligand SYFPEITHI

The proteasome contributes to the generation of most of the peptide ligands of MHC class I molecules. To compare the identity of the peptides generated by the proteasome with those finally presented by MHC class I molecules, we generated a monoclonal antibody recognizing the C‐terminal part of the dominant H2‐Ku2009d ligand SYFPEITHI derived from the JAK1 tyrosine kinase. Immunoprecipitations of lysates from H2‐Ku2009du2009‐expressing or non‐expressing cells revealed that only in the presence of H2‐Ku2009d SYFPEITHI could be isolated. No longer potential precursor peptide containing SYFPEITHI could be detected. Surprisingly, a peptide lacking the first two amino acids, FPEITHI, was isolated independently of the presence of H2‐Ku2009d molecules. The detection of only SYFPEITHI and FPEITHI in cell lysates corresponded with the strong generation of these two peptides in in vitro digests of elongated SYFPEITHI‐containing peptides with purified 20S proteasomes. Our results indicate that MHC ligands can be generated directly by the proteasome in vivo and that at least for SYF PEITHI the expression of the corresponding MHC molecule is critical for protection of the ligand in vivo.

Polymorphism of minor histocompatibility genes in wild mice.

H-2b-restricted cytolytic T lymphocytes (CTL) were generated against H-1, H-3, and H-4 antigens and tested against target cells of F1 hybrids between wild mice and inbred H-2b mice. The congenic strain combinations for the CTL production were such that they tested one allele each at the H-1 and H-4 loci and four alleles at the H-3 locus. Most of the wild mice tested came from Southern Germany, but a few mice came from other European countries and Egypt and Israel. Virtually all wild mice typed as positive with CTL directed against H-3b and H-4b antigens; 32% of the F1 hybrids tested reacted with anti-H-1cCTL and 9% reacted with anti-H-3d CTL. The positive results were not caused by cross-reaction with allogeneic H-2 antigens controlled by the major histocompatibility complex (Mhc) genes of the wild mice. At least some of the H-3 and H-4 antigens detected by the CTL in the F1 hybrid were not identical with antigens of the immunizing strains. These results suggest a relatively low degree of polymorphism of the tested minor H loci in wild mice and further support the notion that minor H loci are unrelated to the Mhc.

Der Haupthistokompatibilittskomplex und die Unterscheidung zwischen Selbst und Fremd durch das Immunsystem

The major histocompatibility complex (Mhc) is a cluster of closely linked genes which are involved in the distinction between self and non-self. The genes fall into two classes, I and II, which are evolutionarily related but specialized to performing somewhat different functions. The Mhc genes code for proteins which are seen together with foreign substances by the thymus-derived lymphocytes. These lymphocytes thus recognize simultaneously self (Mhc molecules) and non-self (foreign antigen). Some of the Mhc genes are highly polymorphic and this polymorphism probably represents a compensation for the fact that certain combinations of Mhc molecules and antigen fail to be recognized by the T lymphocyte.