Molecular definition of a polymorphic antigen (LA45) of free HLA-A and -B heavy chains found on the surfaces of activated B and T cells.

A monomoprhic monoclonal antibody (LA45 antibody) reactive with "a new activation-induced surface structure on human T lymphocytes" (LA45 antigen) that resembled free class I heavy chains has recently been described (Schnabl, E., H. Stockinger, O. Majdic, H. Gaugitsch, I.J.D. Lindley, D. Maurer, A. Hajek-Rosenmayr, and W. Knapp. 1990. J. Exp. Med. 171:1431). This antibody was used to clone a class I-like heavy chain (LA45 gene) from the HUT 102 tumor cell, which paradoxically did not give rise to the LA45 antigen on transfection into monkey COS cells. We show here that the LA45 gene is HLA-Aw66.2, a previously uncharacterized allele of the HLA-A locus. The previously determined LA45 sequence differs from that of HLA-Aw66.2, from HUT 102, and the CR-B B cell line derived from the same individual as HUT 102 by substitution of tryptophan for serine at position 4 in the alpha 1 domain. Transfection of HLA-Aw66.2, and of a mutant of this gene with serine 4 substituted for tryptophan, into a human B cell line (C1R) both resulted in expression of the LA45 epitope. Furthermore, we find expression of the LA45 epitope on Epstein Barr virus-transformed B cell lines as well as lectin-activated T cells, but not on long-term T cell lines or unstimulated peripheral blood T cells. The specificity of the LA45 antibody is polymorphic and the presence of the LA45 epitope is precisely correlated with the sequence arginine, asparagine (RN) at residues 62 and 63 of the helix of the alpha 1 domain. The LA45 epitope is broadly distributed, being associated with half the alleles of both HLA-A and -B loci but none of the HLA-C locus. All the results are consistent with the presence of pools of free HLA-A and -B heavy chains at the surfaces of certain cell types but not others. Such molecules are probably responsible for the HLA-associated class I alloantigens of lectin-activated T cells. We hypothesize the free heavy chains result from dissociation of beta 2-microglobulin from subpopulations of empty HLA-A,B molecules, or molecules with weakly bound peptides, that vary in size depending on cellular activation and peptide supply.

Proteasome components with reciprocal expression to that of the MHC-encoded LMP proteins.

BACKGROUND: Intracellular proteins are processed into small peptides that bind HLA class I molecules of the major histocompatibility complex (MHC) in order to be presented to T lymphocytes. The proteasome, a multi-subunit protease, has recently been implicated in the generation of these peptides. Two genes encoding proteasome subunits, LMP2 and LMP7, are tightly linked to the TAP peptide transport loci in the class II region of the human MHC. Inclusion of the LMP subunits may alter proteasome activity, biasing it towards the production of peptides with carboxyl termini appropriate for binding HLA class I molecules. Nevertheless, mutant cells that lack the LMP genes are able to process and present antigens at the cell surface at similar levels to wild-type cells. These results raise questions about the role of the proteasome, and in particular of the LMP subunits, in antigen processing. RESULTS: We have cloned the genes encoding a new proteasome subunit, MB1, which is closely related to LMP7, and that encoding a second subunit, Delta, which is closely related to LMP2. Expression of the MB1 and delta genes is reciprocal to that of the LMP genes: MB1 and delta are up-regulated in mutant cell lines lacking LMPs and down-regulated in the presence of gamma-interferon. The MB1 and delta genes are found to be located on chromosomes 14 and 17, respectively, raising interesting evolutionary questions about how the LMP genes independently became incorporated into the MHC. CONCLUSIONS: We suggest that the subtle phenotype of LMP-deficient cell lines results from the compensatory expression in these lines of two other proteasome subunits, MB1 and Delta.

NF-kappaB1 p105 negatively regulates TPL-2 MEK kinase activity.

Activation of the oncogenic potential of the MEK kinase TPL-2 (Cot) requires deletion of its C terminus. This mutation also weakens the interaction of TPL-2 with NF-kappaB1 p105 in vitro, although it is unclear whether this is important for the activation of TPL-2 oncogenicity. It is demonstrated here that TPL-2 stability in vivo relies on its high-affinity, stoichiometric association with NF-kappaB1 p105. Formation of this complex occurs as a result of two distinct interactions. The TPL-2 C terminus binds to a region encompassing residues 497 to 534 of p105, whereas the TPL-2 kinase domain interacts with the p105 death domain. Binding to the p105 death domain inhibits TPL-2 MEK kinase activity in vitro, and this inhibition is significantly augmented by concomitant interaction of the TPL-2 C terminus with p105. In cotransfected cells, both interactions are required for inhibition of TPL-2 MEK kinase activity and, consequently, the catalytic activity of a C-terminally truncated oncogenic mutant of TPL-2 is not affected by p105. Thus, in addition to its role as a precursor for p50 and cytoplasmic inhibitor of NF-kappaB, p105 is a negative regulator of TPL-2. Insensitivity of C-terminally truncated TPL-2 to this regulatory mechanism is likely to contribute to its ability to transform cells.

Association of mucosal leishmaniasis with HLA.

In the search for genetic variability in individual susceptibility to mucocutaneous leishmaniasis, a disease caused mainly by Leishmania (Viannia) braziliensis, HLA typing was performed on 43 patients presenting mucosal lesions and 111 matched controls. Antigen specificities of the HLA-A, -B, -C, -DR, and -DQ loci were determined and their frequencies in patients and controls were compared. There was a significant decrease in the frequency of HLA-DR2 [1 out of 38 (2.6%) patients vs. 29 out of 102 (28.4%) controls, corrected p value 0.004, relative risk 0.07, preventive fraction of the total population 0.26] as well as a significant increase of HLA-DQw3 [29 out of 38 (76.3%) patients vs. 43 out of 99 (43.4%) controls, corrected p value 0.006, relative risk 4.2, etiologic fraction of the population 0.58]. These results support participation of HLA class II molecules in individual susceptibility to mucocutaneous leishmaniasis and in the pathogenesis of metastatic, mucosal disease.

Proteasome and class I antigen processing and presentation.

The recent discovery of two proteasome homologous genes, LMP2 and LMP7, in the class II region of the human MHC, has implicated this multi-subunit protease in an early step of the immune response; the degradation of intracellular and viral proteins. Short peptides produced by the proteasome are transported into the ER by the product of another set of MHC class II genes, TAP1 and TAP2, where they bind and stabilise HLA class I molecules. Antigenic peptides displayed at the cell surface by HLA class I molecules mark cells for destruction by cytotoxic T lymphocytes. The role of the proteasome in antigen processing was questioned when mutant cells, which lack the LMP genes, were able to process and present antigens normally. The discovery that two proteasome beta-subunits, delta and MB1, highly homologous to LMP2 and LMP7 and expressed in reciprocal manner, is now consistent with a role for the proteasome in antigen processing. The incorporation of different beta-subunits into the proteasome may be a mechanism to modulate catalytic activity of the proteasome complex, allowing production of peptides that are more suitable to enter into the ER by the TAP transporters and to bind HLA class I molecules. But, in the absence of the LMPs, the other subunits permit processing of most antigens reasonably efficiently.

TPL-2 kinase regulates the proteolysis of the NF-kappaB-inhibitory protein NF-kappaB1 p105.

The transcription factor NF-kappaB is composed of homodimeric and heterodimeric complexes of Rel/NF-kappaB-family polypeptides, which include Rel-A, c-Rel, Rel-B, NF-kappaB/p50 and NF-kappaB2/p52 . The NF-kappaB1 gene encodes a larger precursor protein, p105, from which p50 is produced constitutively by proteasome-mediated removal of the p105 carboxy terminus. The p105 precursor also acts as an NFkappaB-inhibitory protein, retaining associated p50, c-Rel and Rel-A proteins in the cytoplasm through its carboxy terminus. Following cell stimulation by agonists, p105 is proteolysed more rapidly and released Rel subunits translocate into the nucleus. Here we show that TPL-2 , which is homologous to MAP-kinase-kinase kinases in its catalytic domain, forms a complex with the carboxy terminus of p105. TPL-2 was originally identified, in a carboxy-terminal-deleted form, as an oncoprotein in rats and is more than 90% identical to the human oncoprotein COT. Expression of TPL-2 results in phosphorylation and increased degradation of p105 while maintaining p50 production. This releases associated Rel subunits or p50-Rel heterodimers to generate active nuclear NF-kappaB. Furthermore, kinase-inactive TPL-2 blocks the degradation of p105 induced by tumour-necrosis factor-alpha. TPL-2 is therefore a component of a new signalling pathway that controls proteolysis of NF-kappaB1 p105.

Serologic cross-reactivities poorly reflect allelic relationships in the HLA-B12 and HLA-B21 groups. Dominant epitopes of the alpha 2 helix.

Previous analysis has emphasized the correlation between primary structures of class I HLA molecules and their patterns of serologic cross-reactivity. Here we describe the structures of two serologic groups of HLA-B alleles for which this is not the case. HLA-B45, an allele associated with black populations, is serologically paired with B44 in the B12 group; its structure, however, is divergent from that of B44 but closely related to B50. The BN21 (B*4005) allele is associated with native Americans and is serologically grouped with B50 in the B21 group; its structure, however, is more closely related to alleles of the B40 group. The B44 and B45 serologically cross-reactive molecules differ at seven functional positions of the Ag recognition site; the B50 and BN21 molecules differ at four such residues. These differences are predicted to alter peptide presentation and be capable of eliciting strong alloreactive T cell responses. For these pairs of B12 and B21 Ag, serology appears dominated by epitopes formed by short sequences of the alpha 2 helix which have been shuffled by recombination between alleles. The implications of these results for HLA matching in transplantation are discussed.

Unusual HLA-B alleles in two tribes of Brazilian Indians.

The Kaingang and Guarani are culturally and linguistically distinct tribes of southern Brazil. Like all Amerindian groups they show limited HLA polymorphism, which probably reflects the small founder populations that colonized America by overland migration from Asia 11,000-40,000 years ago. We find the nucleotide sequences of HLA-B alleles from the Kaingang and Guarani to be distinct from those characterized in caucasian, oriental and other populations. By comparison, the HLA-A and C alleles are familiar. These results and those reported in the accompanying paper on the Waorani of Ecuador reveal that a marked evolution of HLA-B has occurred since humans first entered South America. New alleles have been formed through recombination between pre-existing alleles, not by point mutation, giving rise to distinctive diversification of HLA-B in different South American Indian tribes.

Structural diversity in the HLA-A10 family of alleles: correlations with serology.

The HLA-A10 crossreacting group consists of the A25, A26, A34, A43 and A66 antigens. Here, we report allelic sequences for A43 and for 2 subtypes of both A26 and A34. Combining these results with previously determined sequences for A25, A26 and A66 enables molecular comparison of all the serologically defined A10 antigens. They form a closely related and well-defined group of alleles which may have originated with A*2601. Patterns of serological crossreactivity are correlated with sequence and a public epitope shared by A33 and members of the A10 family is localized to residues R62 and N63. The A*2501, A*4301 and A*6601 alleles appear to have derived from A*2601 by single gene conversion events with other HLA-A alleles. In the case of A*4301, the donor allele was probably an A29 allele as A*4301 has a small element of sequence in the alpha 1 helix (residues L62 and Q63) uniquely shared with A29. The chimaeric structure of A43 explains the reactivity of A43 molecules with both A10 and A29 alloantisera. The rare Oriental variant of A26 (A26v*) is encoded by an allele (A*2602) that differs from A*2601 by a unique nucleotide substitution which changes aspartate to asparagine at position 116 in the floor of the peptide binding groove. Thus A*2602 is a functionally distinct allele that originated by a point mutation. Alleles encoding A34 and A66 antigens are found to have very similar structures, explaining the difficulty in their serological definition.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinctive HLA-A,B antigens of black populations formed by interallelic conversion.

Alleles encoding five HLA-A and B Ag characteristic of black populations have been isolated and their nucleotide sequences determined. In each case, the "black" allele is similar to a "related" allele found in caucasoid populations. The primary differences between these pairs of alleles are localized clusters of nucleotide substitutions that change two to five residues of the Ag recognition site. The pattern of differences indicates that the pairs of black and caucasoid alleles diverged primarily as a result of interallelic conversion events.