Nick Holmes

Nonclassical HLA-G molecules are classical peptide presenters

BACKGROUND The physiological functions of the classical HLA (human leukocyte antigen) molecules, HLA-A, HLA-B and HLA-C, are to present peptides to T cells and to inhibit the activity of natural killer cells. In contrast, the functions of nonclassical HLA-molecules, such as HLA-E, HLA-F and HLA-G, remain to be established. The expression of HLA-G is largely limited to the placental trophoblast, where it might mediate protection of the fetus from rejection by the mother. Achieving the aim of understanding the function of HLA-G should be facilitated by information on the biochemical properties of HLA-G molecules, especially on their potential ability to act as peptide receptors. RESULTS To study peptide presentation by HLA-G, we used stably transfected LCL721.221 cells as a source of HLA-G molecules and analysed the spectrum of extracted peptides by individual and pool sequencing. Our results indicate that HLA-G molecules, like classical HLA molecules, are associated with a wide array of peptides derived from cellular proteins. Peptides presented by HLA-G usually consisted of 9 amino acids, and adhered to a specific sequence motif, with anchor residues at position 2 (isoleucine or leucine), position 3 (proline) and the carboxy-terminal position 9 (leucine). Thus, the HLA-G peptide ligand motif follows the principles of classical HLA motifs, although it displays its own unique features. Peptide-binding assays indicated that two of the three anchor residues were sufficient for binding, and that the three natural HLA-G ligands that we identified bound, not only to HLA-G, but also to HLA-A2. This was not surprising, because the binding pockets of HLA-A2 and HLA-G overlap in their ability to recognize anchor residues at positions 2 and 9. Likewise, some, but not all, HLA-A2 peptide ligands could also bind to HLA-G. CONCLUSIONS Nonclassical HLA-G molecules present peptides essentially in the same way as classical HLA molecules do. We determined the peptide motif that is specifically recognized by HLA-G; its basic features are described by the sequence XI/LPXXXXXL: This information should help to elucidate the physiological role of HLA-G molecules at the fetal-maternal interface. Most likely, this role is to protect fetal cells from lysis by natural killer cells, and possibly to present foreign peptides to a class of T cells that has not yet been identified.

A homodimeric complex of HLA-G on normal trophoblast cells modulates antigen-presenting cells via LILRB1.

In healthy individuals, the non‐classical MHC molecule HLA‐G is only expressed on fetal trophoblast cells that invade the decidua during placentation. We show that a significant proportion of HLA‐G at the surface of normal human trophoblast cells is present as a disulphide‐linked homodimer of the conventional β2m‐associated HLA‐I complex. HLA‐G is a ligand for leukocyte immunoglobulin‐like receptors (LILR), which bind much more efficiently to dimeric HLA‐G than to conventional HLA‐I molecules. We find that a LILRB1‐Fc fusion protein preferentially binds the dimeric form of HLA‐G on trophoblast cells. We detect LILRB1 expression on decidual myelomonocytic cells; therefore, trophoblast HLA‐G may modulate the function of these cells. Co‐culture with HLA‐G+ cells does not inhibit monocyte‐derived dendritic cell up‐regulation of HLA‐DR and costimulatory molecules on maturation, but did increase production of IL‐6 and IL‐10. Furthermore, proliferation of allogeneic lymphocytes was inhibited by HLA‐G binding to LILRB1/2 on responding antigen‐presenting cells (APC). As HLA‐G is the only HLA‐I molecule that forms β2m‐associated dimers with increased avidity for LILRB1, this interaction could represent a placental‐specific signal to decidual APC. We suggest that the placenta is modulating maternal immune responses locally in the uterus through HLA‐G, a trophoblast‐specific, monomorphic signal present in almost every pregnancy.

In situ hybridization and northern blot demonstration of HLA-G mRNA in human trophoblast populations by locus-specific oligonucleotide

Trophoblast cells from normal first trimester pregnancies have been shown to express the nonclassical class I molecule, HLA-G, which is nonpolymorphic and has a heavy chain of 40 kD. In this study, in situ hybridization experiments were performed using a biotinylated HLA-G specific oligonucleotide on sections of normal placenta including the implantation site. HLA-G mRNA was identified in all extravillous trophoblast populations including the cytotrophoblast cell columns, interstitial trophoblast, endovascular trophoblast, and placental bed giant cells. In addition, villous cytotrophoblast and villous mesenchymal cells also contained HLA-G transcripts, but villous syncytiotrophoblast was always negative.

CD45: all is not yet crystal clear

CD45 has been recognized as an important player in regulating signalling in lymphocytes. However, compared with tyrosine kinases, phosphatases are still poorly understood in terms of the details of their specificity and regulation. Here, the recent progress in understanding the biology of the first recognized receptor tyrosine phosphatase, CD45, is reviewed.

Generation of an antibody to HLA-G in transgenic mice and demonstration of the tissue reactivity of this antibody.

A method was devised to generate antibodies against the non-classical class I HLA-G antigen. This consisted of immunising HLA-A2/beta 2m double transgenic mice with HLA-G transfected into mouse Ltk- cells. A polyclonal antibody was obtained which appears to be specific for HLA-G. The staining pattern of this antibody was restricted solely to all populations of extravillous trophoblast. No fetal tissues reacted with this antibody, including those where HLA-G mRNA has been demonstrated, such as fetal eye, thymus and liver. This study confirms that HLA-G is a trophoblast-specific protein, although it remains a possibility that the technique of immunohistology is not sufficiently sensitive to detect low level HLA-G antigen expression in non-trophoblast tissues.

Either of the CD45RB and CD45RO Isoforms Are Effective in Restoring T Cell, But Not B Cell, Development and Function in CD45-Null Mice

The protein tyrosine phosphatase CD45 is expressed as a series of isoforms whose tissue and differentiation stage specificity is broadly conserved in evolution. CD45 has been shown to be an important regulator of a variety of functions in many different hemopoietic lineages. We have chosen an in vivo genetic complementation strategy to investigate the differential functions between isoforms. In this study, we report the characterization of transgenic mice which express the isoforms CD45RO or CD45RB as their only CD45 molecules, at a variety of expression levels and in the majority of hemopoietic lineages. Both CD45RO and CD45RB isoforms reconstitute thymocyte development in a CD45-null mouse background when expressed above a threshold level. The resulting mature T cells populate the peripheral lymphoid organs where they are found at normal frequency. Both CD45RO and CD45RB isoforms also permit T cell function in the periphery, although the threshold for normal function here appears to be set higher than in the thymus. In contrast, neither isoform is capable of fully restoring peripheral B cell maturation, even at levels approaching those in heterozygous CD45+/− mice in which maturation is normal. In vitro activation of B cells by Ag-receptor stimulation is only minimally complemented by these CD45RO and CD45RB transgenes. Our results suggest that CD45 isoforms play unique roles which differ between the T and B lineages.

The CD45 tyrosine phosphatase regulates CD3‐induced signal transduction and T cell development in recombinase‐deficient mice: restoration of pre‐TCR function by active p56lck

The pre‐TCR complex regulates the transition from CD4–CD8– double‐negative (DN) to CD4+CD8+ double‐positive (DP) thymocytes during T cell development. In CD45– / – mice there is an accumulation of DN cells, suggesting a possible role for CD45 in pre‐TCR signaling. We therefore crossed CD45– / – with Rag‐1– / – mice to investigate the signaling functions of the CD3 complex in DN thymocytes. Remarkably, treatment of Rag‐1– / – / CD45– / – mice with a CD3 mAb caused maturation to the DP stage at only 3 % of the level measured in Rag‐1– / – mice. Furthermore, ligation of the CD3 complex on Rag‐1– / – / CD45– / – thymocytes in vitro induced less tyrosine phosphorylation in specific proteins when compared to Rag‐1– / – thymocytes. CD45– / – mice were also crossed with pLGFA mice expressing a constitutively active form of the lck tyrosine kinase which restored the DN to DP transition to near normal levels. Our results are consistent with a model in which CD45‐activated p56lck is critical for pre‐TCR signal transduction.

Defective depletion of CD45-null thymocytes by the Staphylococcus aureus Enterotoxin B superantigen

The development of a normal T-cell repertoire is critically dependent on the negative and positive selection events which occur at the CD4+CD8+ (double positive, DP) stage of thymic development. Depending on the avidity of the T-cell antigen receptor (TCR) for peptides presented within the thymus, DP thymocytes are either positively selected for maturation to CD4+/CD8+ single positive cells or are depleted by apoptosis. The addition of superantigen to thymocytes within foetal thymic organ culture (FTOC) mimics the negative selection signal of potentially autoreactive thymocytes and induces the responding population of thymocytes to apoptose. Here we present evidence that the transmembrane phosphotyrosine phosphatase CD45 critically regulates TCR-induced signals in thymic differentiation and present data to show defective depletion of CD45-null transgenic TCR-Vbeta8 DP thymocytes in FTOC by the Staphylococcus aureus Enterotoxin B (SEB) superantigen.

An oriental HLA-A2 subtype is closely related to a subset of caucasoid HLA-A2 alleles

An important property of the genes encoding the class I glycoproteins of the mammalian major histocompatibility complex (MHC) is their high degree of polymorphism in most species. The mechanisms involved in generation of variation in the DNA sequences of these genes and the means by which new alleles increase in frequency and are maintained in the population are therefore of considerable interest. We have chosen to approach the first of these topics by studying the human class I genes, in particular the HLA-A locus. Serological analyses at a series of international workshops have resulted in the identification of 20 different specificities or serotypes at the HLA-A locus. By far the most frequent HLA-A locus specificity is HLAA2, which is found in about 50% of Caucasoids. The standard method used for HLA typing is a complement dependent cytotoxicity test employing alloantisera and monoclonal antibodies (mAb). The HLA-A2 antigen defined by these means actually consists of a family of many closely related alleles. The various HLA-A2 allelic products can be distinguished from one another by cytotoxic T cell recognition (Biddison et al. 1980, Gaston et al. 1983, van der Poel et al. 1983a, Gotch et al. 1985) and in some cases by monoclonal antibodies (Russo et al. 1983, Fauchet et al. 1984, Kennedy et al. 1987), and/or by isoelectric focusing (Biddison et al. 1982, van der Poel et al. 1983b). Eight of these alleles have been previously characterized at the level of primary sequence and their names are listed in Table 1. For the sake of consistency and clarity we use the previously described names for these alleles (references given in Table 1); however, new names were given to three of them at the 10th Workshop (Proceedings yet to be published), and these are also listed in Table 1. Any two of the HLA-A2 genes differ at no more than six amino acids, and between some variants only a

A SNP in the immunoregulatory molecule CTLA-4 controls mRNA splicing in vivo but does not alter diabetes susceptibility in the NOD mouse

CTLA-4 is a critical “checkpoint” regulator in autoimmunity. Variation in CTLA-4 isoform expression has been linked to type 1 diabetes development in human and NOD mouse studies. In the NOD mouse, a causative link between increased expression of the minor isoform ligand-independent CTLA-4 and a reduction in diabetes has become widely accepted. Altered splicing of CTLA-4 has been attributed to a single nucleotide polymorphism (SNP) in Ctla4 exon2 (e2_77A/G). To investigate this link, we have used NOD embryonic stem (ES) cells to generate a novel NOD transgenic line with the 77A/G SNP. This strain phenocopies the increase in splicing toward the liCTLA4 isoform seen in B10 Idd5.1 mice. Crucially, the SNP does not alter the spontaneous incidence of diabetes, the incidence of cyclophosphamide-induced diabetes, or the activation of diabetogenic T-cell receptor transgenic CD4+ T cells after adoptive transfer. Our results show that one or more of the many other linked genetic variants between the B10 and NOD genome are required for the diabetes protection conferred by Idd5.1. With the NOD mouse model closely mimicking the human disease, our data demonstrate that knock-in transgenic mice on the NOD background can test causative mutations relevant in human diabetes.