Maria J. Recio

Differential Biological Role of CD3 Chains Revealed by Human Immunodeficiencies

The biological role in vivo of the homologous CD3γ and δ invariant chains within the human TCR/CD3 complex is a matter of debate, as murine models do not recapitulate human immunodeficiencies. We have characterized, in a Turkish family, two new patients with complete CD3γ deficiency and SCID symptoms and compared them with three CD3γ-deficient individuals belonging to two families from Turkey and Spain. All tested patients shared similar immunological features such as a partial TCR/CD3 expression defect, mild αβ and γδ T lymphocytopenia, poor in vitro proliferative responses to Ags and mitogens at diagnosis, and very low TCR rearrangement excision circles and CD45RA+ αβ T cells. However, intrafamilial and interfamilial clinical variability was observed in patients carrying the same CD3G mutations. Two reached the second or third decade in healthy conditions, whereas the other three showed lethal SCID features with enteropathy early in life. In contrast, all reported human complete CD3δ (or CD3ε) deficiencies are in infants with life-threatening SCID and very severe αβ and γδ T lymphocytopenia. Thus, the peripheral T lymphocyte pool was comparatively well preserved in human CD3γ deficiencies despite poor thymus output or clinical outcome. We propose a CD3δ ≫ CD3γ hierarchy for the relative impact of their absence on the signaling for T cell production in humans.

TRANSCRIPTION AND WEAK EXPRESSION OF HLA-DRB6 : A GENE WITH ANOMALIES IN EXON 1 AND OTHER REGIONS

Abstract HLA-DRB6 is one of the human major histocompatibility complex (MHC) genes present in DR1, DR2, and DR10 haplotypes (approximately 26% of individuals). It shows several anomalies in human and non-human primates, including exon 2 stop codons (non-randomly grouped between codons 74 and 94) and a promoter region, and an exon 1 coming from an inserted retrovirus. It has been shown that not only chimpanzee but also human Mhc-DRB6 lack the usual 3’ untranslated (UT) polyadenylation signal, and in the present work it was found that the human DRB6 gene coming from an HLA-DR2 haplotype is effectively transcribed after transfection in mouse L cells, and that HLA-DRB6 molecules may be expressed on the cell surface. DRB6 transcription level is remarkably lower in human than in chimpanzee. Moreover, their exons 1 (both taken from the 3’LTR region of a mammary tumor retrovirus) are also different; this shows that these viral insertions may be an important mechanism for different evolutionary changes in orthologous genes of different species. The pathways by which DRB6 molecules may be expressed on the membrane are unclear but other examples of truncated protein expression have also been described, even within the human major histocompatibility complex (i. e., in HLA-G). Finally, the presence of mature HLA-DRB6 mRNA molecules supports the notion that splicing may take place even in the absence of a canonical 3’UT polyadenylation signal.

Human CD3γ, but not CD3δ, haploinsufficiency differentially impairs γδ versus αβ surface TCR expression

BackgroundThe T cell antigen receptors (TCR) of αβ and γδ T lymphocytes are believed to assemble in a similar fashion in humans. Firstly, αβ or γδ TCR chains incorporate a CD3δε dimer, then a CD3γε dimer and finally a ζζ homodimer, resulting in TCR complexes with the same CD3 dimer stoichiometry. Partial reduction in the expression of the highly homologous CD3γ and CD3δ proteins would thus be expected to have a similar impact in the assembly and surface expression of both TCR isotypes. To test this hypothesis, we compared the surface TCR expression of primary αβ and γδ T cells from healthy donors carrying a single null or leaky mutation in CD3G (γ+/−) or CD3D (δ+/−, δ+/leaky) with that of normal controls.ResultsAlthough the partial reduction in the intracellular availability of CD3γ or CD3δ proteins was comparable as a consequence of the mutations, surface TCR expression measured with anti-CD3ε antibodies was significantly more decreased in γδ than in αβ T lymphocytes in CD3γ+/− individuals, whereas CD3δ+/− and CD3δ+/leaky donors showed a similar decrease of surface TCR in both T cell lineages. Therefore, surface γδ TCR expression was more dependent on available CD3γ than surface αβ TCR expression.ConclusionsThe results support the existence of differential structural constraints in the two human TCR isotypes regarding the incorporation of CD3γε and CD3δε dimers, as revealed by their discordant surface expression behaviour when confronted with reduced amounts of CD3γ, but not of the homologous CD3δ chain. A modified version of the prevailing TCR assembly model is proposed to accommodate these new data.

A new sequence (Mhc-BJ ) showing similarity both to Mhc-B alleles and to the HLA-J pseudogene in Macaca mulatta

Class I major histocompatibility complex (Mhc) genes in humans have been divided into two groups: 1) classical (HLA-A, -B, and -C) that encode polymorphic membrane glycoproteins implicated in the presentation of intracellular derived peptides to the cytotoxic T cells, and 2) nonclassical that include a group of genes (HLA-E, -F, and -G) which code for molecules whose functions have not yet been defined (and certainly are not antigen presenting molecules in the case of -G and Macaca mulatta family; Castro et al. 1996) and also a set of several pseudogenes [(HLA-H, -J, -K, -L) (Bodmer et al. 1995)]. The mechanisms implicated in generating antigen site diversity of the presenting Mhc molecules remain unclear. It has been suggested that this diversity is generated by point mutation, segmental exchange, and recombination events, and then selection maintains or deletes the new products generated in a population (Klein et al. 1990). An Mhc class I sequencing study in M. mulatta (rhesus monkeys) was carried out to define possible new alleles or genes and investigate the mechanisms responsible for the generation of diversity of the Mhc alleles in apes. A new Mhc class I sequence was found, characterized by a 75 nucleotide deletion at the beginning of exon 3, which resembled Mhc-B alleles and possibly HLA-J sequences. Whole RNA was extracted from kidney cell lines (107 cells per line) for two M. mulatta individuals (rhesus monkeys, Rh-8, Rh-14) using NP-40 standard protocol. cDNA synthesis was performed using a reverse transcription system (Promega, Madison, WI) according to the manufacturer’s protocols. Partial exon 1, exon 2, exon 3 and partial exon 4 from this new Mamu-Mhc nucleotide sequence gene was obtained by polymerase chain reaction (PCR) using 5E1m (59-CGCTCCTCCTGCTGCTCTCGGC) and 3E4m (59-AGATGGGGTGGTGGGTCACG) primers. Random sequencing of the amplified products was carried out and PCR products were also purified and inserted into the pGEM-T vector (Promega). Doublestranded DNA stretches were automatically sequenced in an Applied Biosystems (Foster City, CA) machine as previously described (Castro et al. 1996). Exonic phylogenetic trees were made using the neighbor-joining method (Saitou and Nei) and the computer program NJDRAW, designed by J. W. H. Ferguson. A DNA phylogenetic tree was based on the number of nucleotide substitutions per site, and was corrected for multiple hits by the Jukes and Cantor (1969) method using the computer program NAG (version 2.0), designed by T. Ota and M. Nei. Five clones cDNA sequences obtained from two different M. mulatta (Rh-8 and Rh-14) were identical and corresponded to this new sequence, previously not found in M. mulatta (Boyson et al. 1995; Castro et al. 1996; Fig. 1). A comparison of its exons with those of classical and non-classical class I alleles showed that exon 2 is more similar to HLA-J and HLA-G*0101 and Mamu-Mhc-G*I and exon 3 is closer to Mamu-Mhc-B*07. These data suggested that this new sequence could be generated by a recombination event between a Mamu-Mhc-B gene and an analogue of HLA-J (see below) postulating a segmental exchange between neighboring genes. It has been tentatively named Mamu-Mhc-BJ and is not a PCR artefact, since it comes from two different individuals, five clones, and different PCR reactions; it has a 75 nucleotide deletion at the begining of exon 3, which would not change the reading frame. A possible protein resulting from a hypothetical transcription and translation would not bear residues Tyr 96 and Tyr 101, which are necessary for the inderdomain dysulphide bond and also lack residues between positions 91– 116 that are implicated on the overall interdomain contacts between α2 and α1 domain and β2-microglobulin (Bjorkman et al. 1987). Thus, a putative MamuMhc-BJ protein, if translated, would not be an antigen The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accesion number L41828 (Mamu-Mhc-BJ)

CD3γ-independent pathways in TCR-mediated signaling in mature T and iNKT lymphocytes

Antigen recognition by T-lymphocytes through the T-cell antigen receptor, TCR-CD3, is a central event in the initiation of an immune response. CD3 proteins may have redundant as well as specific contributions to the intracellular propagation of TCR-mediated signals. However, to date, the relative role that each CD3 chain plays in signaling is controversial. In order to examine the roles of CD3γ chain in TCR signaling, we analyzed proximal and distal signaling events in human CD3γ(-/-) primary and Herpesvirus saimiri (HVS)-transformed T cells. Following TCR-CD3 engagement, certain early TCR signaling pathways (ZAP-70, ERK, p38 and mTORC2 phosphorylation, and actin polymerization) were comparable with control HVS-transformed T cells. However, other signaling pathways were affected, such TCRζ phosphorylation, indicating that the CD3γ chain contributes to improve TCR signaling efficiency and survival. On the other hand, CD3γ(-/-) primary invariant NKT cells (iNKT cells) showed a normal expansion in response to alpha-galactosylceramide (α-GalCer) and TCRVβ11(bright) iNKT cells were preferentially selected in this in vitro culture system, perhaps as a consequence of selective events in the thymus. Our results collectively indicate that a TCR lacking CD3γ can propagate a number of signals through the remaining invariant chains, likely the homologous CD3δ chain, which replaces it at the mutant TCR.

Primary T-cell immunodeficiency with functional revertant somatic mosaicism in CD247

To the Editor: T lymphocytes detect antigens with the T-cell receptor (TCR) composed of a variable heterodimer (either ab or gd), 2 invariant heterodimers (CD3gε and CD3dε), and an invariant homodimer (CD247 or zz). Because of the crucial role of TCR signaling in thymic selection, mutations in TCR, CD3, or CD247 selectively impair T-cell development, albeit to different degrees: deficiency of CD3d or CD3ε, but not of CD3g or CD247, causes severe T-cell lymphopenia. Their clinical outcome is also disparate, because CD3g deficiency does not require urgent transplantation. Thus, TCR immunodeficiencies display a range of phenotypes and careful differential diagnosis is essential for appropriate therapy. We describe an infant born to consanguineous parents with early-onset chronic cytomegalovirus infection, severe immunodeficiency, and extremely low surface TCR levels. Her immunologic characterization at age 11 months is summarized in Table E1 in this article’s Online Repository at www.jacionline.

HLA-DMB in Amerindians: Specific linkage of DMB*01:03:01/DRB1 alleles

BACKGROUND HLA-DMB proteins are important for intracellular microbial metabolism in order other major histocompatibility complex (MHC) molecules present peptides to lymphocytes. In addition, HLA-DMB alleles have been found linked to diseases in some ethnic groups and HLA-DMB molecules may be important to explain HLA disease association. OBJECTIVE To detect HLA-DMB alleles profile in Amerindians for the first time and compare them to other populations. This will establish the bases to study HLA-DMB linkage to disease in Amerindians. METHOD A group of 168 voluntary Amerindians have been typed for HLA-DMB alleles. They have been characterized both, by genetic and genealogical bases. Cloning and automated HLA-DMB DNA (exons 2, 3 and 4) sequencing have been performed for allele assignation. RESULTS HLA-DMB*01:01:01 and HLA-DMB*01:03:01 show the highest frequencies. These have been compared to other World wide populations. HLA-DMB*01:03:01 is tightly associated to certain specific HLA-DRB1 alleles in Amerindians. CONCLUSION The specific Amerindian HLA-DMB allele frequencies and their linkage disequilibrium with other MHC alleles may be crucial to determine HLA-DMB World wide variation, evolution and specific linkage to disease in Amerindians and other populations.

Allelic diversity at the primate Mhc-G locus: Exon 3 bears stop codons in all Cercophitecinae sequences

Twenty-seven major histocompatibility complex (Mhc)-G exon 2, exon 3, and exon 2 and 3 allelic sequences were obtained together with 12 different intron 2 sequences. Homo sapiens, Pan troglodytes, Pan paniscus, Gorilla gorilla, Pongo pygmaeus, Macaca fascicularis, Macaca mulatta, and Cercopithecus aethiops individuals were studied. Polymorphism does not follow the classical pattern of three hypervariable regions per domain and is found in all species studied; exon 3 (equivalent to the alpha 2 protein domain) shows stop codons in the Cercopithecinae group but not in the Pongidae and human groups. Dendrograms show that cotton top tamarin (Saguinus oedipus) Mhc-G sequences are closer to Homo sapiens and Pongidae than to Cercopithecinae, probably due to the stop codons existing at exon 3 of the latter. There is a clear trans-species evolution of allelism in Cercopithecinae and also in exon 2 of all the other apes studied, but a generation of allelism within each species may be present on exon 3 sequences. This discrepancy may be due to the preferential use of exon 2 over exon 3 at the mRNA splicing level within each species in order to obtain the appropriate functional G product. Mhc-G intron 2 shows conserved motifs in all species studied, particularly a 23 base pair deletion between positions 161 and 183 which is locus specific, and some of the invariant residues, important for peptide presentation, conserved in classical class I molecules from fish and reptiles to humans were not found in Mhc-G alleles; the intron 2 dendrogram also shows a particular pattern of allelism within each species. In summary, Mhc-G has substantial differences from other classical class I genes: polymorphism patterns, tissue distribution, gene structure, splicing variability, and probably an allelism variability within each species at exon 3. The G proteins may also be different. This indicates that the Mhc-G function may not be peptide presentation to the clonotypic T-cell receptor.