David A. Rhodes

Relationship between SPRY and B30.2 protein domains. Evolution of a component of immune defence

SPRY and B30.2 are homologous domains which can be identified in 11 protein families encoded in the human genome. These include cell surface receptors of the immunoglobulin super‐family (BTNs), negative regulators of the JAK/STAT pathway (SOCS‐box SSB1–4) and proteins encoded by the numerous TRIM genes. Collectively, proteins containing SPRY and B30.2 domains cover a wide range of functions, including regulation of cytokine signalling (SOCS), RNA metabolism (DDX1, hnRNPs), intracellular calcium release (RyR receptors), immunity to retroviruses (TRIM5alpha) as well as regulatory and developmental processes (HERC1, Ash2L). In order to clarify the evolutionary relationship between the two domains, we compiled a curated database of SPRY and B30.2‐domain sequences. We show that while SPRY domains are evolutionarily ancient, B30.2 domains, found in BTN and TRIM proteins, are a more recent evolutionary adaptation, comprising the combination of SPRY with an additional domain, PRY. The combination of SPRY and PRY to produce B30.2 domains may have been selected and maintained as a component of immune defence.

BTL-II: A POLYMORPHIC LOCUS WITH HOMOLOGY TO THE BUTYROPHILIN GENE FAMILY, LOCATED AT THE BORDER OF THE MAJOR HISTOCOMPATIBILITY COMPLEX CLASS II AND CLASS III REGIONS IN HUMAN AND MOUSE

Abstract Comparison of human and mouse genomic sequence at the border of the major histocompatibility complex (MHC) class II and class III regions revealed a locus encoding six exons with homology to the butyrophilin gene family and the location of a previously decribed gene, testis-specific basic protein (TSBP). We named the new locus BTL-II, for butyrophilin-like MHC class II associated. The six discernable exons of the BTL-II locus encode a small hydrophobic amino acid sequence (which may be a signal peptide), two immunoglobulin domains, a small 7-amino acid, heptad repeat-like exon, and a further two immunoglobulin domains. In mouse, an additional butyrophilin-like gene (NG10) is situated adjacent to BTL-II. Expression studies of the BTL-II locus in mouse showed that it is expressed in a range of gut tissues. We demonstrate that like many other genes from the MHC, BTL-II is polymorphic in a selection of diverse HLA haplotypes. In the light of the newly discovered locus, we revisit and discuss the possible origin of the butyrophilin gene family

BTN1A1, the mammary gland butyrophilin, and BTN2A2 are both inhibitors of T cell activation.

Butyrophilin (BTN) genes encode a set of related proteins. Studies in mice have shown that one of these, BTN1A1, is required for milk lipid secretion in lactation, whereas butyrophilin-like 2 is a coinhibitor of T cell activation. To understand these disparate roles of BTNs, we first compared the expression and functions of mouse Btn1a1 and Btn2a2. Btn1a1 transcripts were not restricted to lactating mammary tissue but were also found in virgin mammary tissue and, interestingly, spleen and thymus. In confirmation of this, BTN1A1 protein was detected in thymic epithelial cells. By contrast, Btn2a2 transcripts and protein were broadly expressed. Cell surface BTN2A2 protein, such as the B7 family molecule programmed death ligand 1, was upregulated upon activation of T cells. We next examined the potential of both BTN1A1 and BTN2A2 to interact with T cells. Recombinant Fc fusion proteins of murine BTN2A2 and, surprisingly BTN1A1, bound to activated T cells, suggesting the presence of one or more receptors on these cells. Immobilized BTN-Fc fusion proteins, but not MOG-Fc protein, inhibited the proliferation of CD4 and CD8 T cells activated by anti-CD3. BTN1A1 and BTN2A2 also inhibited T cell metabolism, IL-2, and IFN-γ secretion. Inhibition of proliferation was not abrogated by exogenous IL-2 but could be overcome following costimulation with high levels of anti-CD28 Ab. These data are consistent with a coinhibitory role for mouse BTNs, including BTN1A1, the BTN expressed in the lactating mammary gland and on milk lipid droplets.

Activation of Human γδ T Cells by Cytosolic Interactions of BTN3A1 with Soluble Phosphoantigens and the Cytoskeletal Adaptor Periplakin

The three butyrophilin BTN3A molecules, BTN3A1, BTN3A2, and BTN3A3, are members of the B7/butyrophilin-like group of Ig superfamily receptors, which modulate the function of T cells. BTN3A1 controls activation of human Vγ9/Vδ2 T cells by direct or indirect presentation of self and nonself phosphoantigens (pAg). We show that the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate binds to the intracellular B30.2 domain of BTN3A1 with an affinity of 1.1 μM, whereas the endogenous pAg isopentenyl pyrophosphate binds with an affinity of 627 μM. Coculture experiments using knockdown cell lines showed that in addition to BTN3A1, BTN3A2 and BTN3A3 transmit activation signals to human γδ T cells in response to (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and the aminobisphosphonate drug zoledronate that causes intracellular accumulation of isopentenyl pyrophosphate. The plakin family member periplakin, identified in yeast two-hybrid assays, interacted with a membrane-proximal di-leucine motif, located proximal to the B30.2 domain in the BTN3A1 cytoplasmic tail. Periplakin did not interact with BTN3A2 or BTN3A3, which do not contain the di-leucine motif. Re-expression into a BTN3A1 knockdown line of wild-type BTN3A1, but not of a variant lacking the periplakin binding motif, BTN3A1Δexon5, restored γδ T cell responses, demonstrating a functional role for periplakin interaction. These data, together with the widespread expression in epithelial cells, tumor tissues, and macrophages detected using BTN3A antiserum, are consistent with complex functions for BTN3A molecules in tissue immune surveillance and infection, linking the cell cytoskeleton to γδ T cell activation by indirectly presenting pAg to the Vγ9/Vδ2 TCR.

Assignment of the human ferrochelatase gene (FECH) and a locus for protoporphyria to chromosome 18q22

We have mapped the human gene for ferrochelatase (FECH; ferroheme-protolyase, EC 4.99.1.1) to chromosome 18 by hybridization of cDNA to sorted chromosomes. The probe was obtained by PCR-directed amplification of a human marrow cDNA library in lambda gt 10. Subchromosomal localization of ferrochelatase to 18q22 was determined by chromosomal hybridization in situ using a human ferrochelatase genomic clone in lambda EMBL 3 that contained a 20-kb insert. Since ferrochelatase activity is deficient in patients with the inherited disease erythropoietic protoporphyria, a locus for this disease may be assigned to 18q22, one of few monogenic defects that have been mapped to this chromosome.

Intracellular antibody-mediated immunity and the role of TRIM21

Protection against bacterial and viral pathogens by antibodies has always been thought to end at the cell surface. Once inside the cell, a pathogen was understood to be safe from humoral immunity. However, it has now been found that antibodies can routinely enter cells attached to viral particles and mediate an intracellular immune response. Antibody‐coated virions are detected inside the cell by means of an intracellular antibody receptor, TRIM21, which directs their degradation by recruitment of the ubiquitin‐proteasome system. In this article we assess how this discovery alters our view of the way in which antibodies neutralise viral infection. We also consider the antiviral function of TRIM21 in the context of its other reported roles in immune signalling and autoimmunity. Finally, we discuss the conceptual implications of intracellular antibody immunity and how it alters our view of the discrete separation of extracellular and intracellular environments.

The B7 Homolog Butyrophilin BTN2A1 Is a Novel Ligand for DC-SIGN

The MHC-encoded butyrophilin, BTN2A1, is a cell surface glycoprotein related to the extended family of B7 costimulatory molecules. BTN2A1 mRNA was expressed in most human tissues, but protein expression was significantly lower in leukocytes. An Ig-fusion protein of BTN2A1 bound to immature monocyte-derived dendritic cells. Binding diminished upon MoDC maturation and no binding was detected to Langerhans cells. Induction of the counterreceptor was IL-4 dependent and occurred early during dendritic cell differentiation. The interaction required the presence of Ca2+ and was mediated by high-mannose oligosaccharides. These properties matched DC-SIGN, a DC-specific HIV-1 entry receptor. This was confirmed by binding of soluble BTN2A1 to DC-SIGN-transfectants and its inhibition by a specific Ab. DC-SIGN bound to native BTN2A1 expressed on a range of tissues. However, BTN2A1 was not recognized on some normal cells such as HUVECs despite a similar expression level. The BTN2A1 of tumor cells such as HEK293T have more high-mannose moieties in comparison to HUVECs, and those high-mannose moieties are instrumental for binding to DC-SIGN. The data are consistent with tumor- or tissue-specific glycosylation of BTN2A1 governing recognition by DC-SIGN on immature monocyte-derived dendritic cells.

Regulation of Immunity by Butyrophilins.

Butyrophilin molecules (commonly contracted to BTN), collectively take their name from the eponymous protein in cows milk. They are considered to be members of the B7 family of costimulatory receptors, which includes B7.1 (CD80), B7.2 (CD86), and related molecules, such as PD-L1 (B7-H1, CD274), ICOS-L (CD275), and B7-H3 (CD276). These coreceptors modulate T cell responses upon antigen presentation by major histocompatibility complex and cognate αβ T cell receptor engagement. Molecules such as BTN3A1 (CD277), myelin oligodendrocyte glycoprotein, and mouse Skint1 and Btnl2, all members of the butyrophilin family, show greater structural and functional diversity than the canonical B7 receptors. Some butyrophilins mediate complex interactions between antigen-presenting cells and conventional αβ T cells, and others regulate the immune responses of specific γδ T cell subsets by mechanisms that have characteristics of both innate and adaptive immunity.

TRIM21 and the Function of Antibodies inside Cells

Therapeutic antibodies targeting disease-associated antigens are key tools in the treatment of cancer and autoimmunity. So far, therapeutic antibodies have targeted antigens that are, or are presumed to be, extracellular. A largely overlooked property of antibodies is their functional activity inside cells. The diverse literature dealing with intracellular antibodies emerged historically from studies of the properties of some autoantibodies. The identification of tripartite motif (TRIM) 21 as an intracellular Fc receptor linking cytosolic antibody recognition to the ubiquitin proteasome system brings this research into sharper focus. We review critically the research related to intracellular antibodies, link this to the TRIM21 effector mechanism, and highlight how this work is exposing the previously restricted intracellular space to the potential of therapeutic antibodies.

Detection of hereditary haemochromatosis in an HLA-identical pedigree showing discordance between HLA class I genes and the disease locus

SummaryHereditary haemochromatosis is a recessive disease in which primary hepatocellular carcinoma, complicating cirrhosis, is responsible for about one-third of deaths in affected homozygotes. We describe a unique HLA haplo-identical pedigree showing parent-to-off-spring transmission of hereditary haemochromatosis in whom HLA typing studies, including class I and class II allogenotype analysis, were of no benefit in identifying affected homozygotes. However, affected siblings in the pre-cirrhotic stage of haemochromatosis, with apparent discordance between the haemochromatosis allele and class I loci on chromosome 6, were detected by undertaking a family study, using analysis of serum parameters of iron status in combination with magnetic resonance imaging (MRI). This pedigree emphasises the critical importance of genetic and non-invasive methods for the identification of asymptomatic homozygotes before cirrhosis develops.