REVIEWS

Immune modulation by butyrophilins Heather A. Arnett1 and Joanne L. Viney2

Abstract | The B7 family of co‑stimulatory molecules has an important role in driving the activation and inhibition of immune cells. Evolving data have shown that a related family of molecules — the butyrophilins — have similar immunomodulatory functions to B7 family members and may represent a novel subset of co‑stimulatory molecules. These studies have taken the field by surprise, as the butyrophilins were previously thought to only be important in lactation and milk production. In this Review, we describe the expression patterns of the various members of the butyrophilin family and explore their immunomodulatory functions. In particular, we emphasize the contribution of butyrophilins to immune homeostasis and discuss the potential of targeting these molecules for therapeutic purposes.

Department of Inflammation, Amgen, Seattle, Washington, USA. 2 Department of Immunology, Biogen Idec, Cambridge, Massachusetts, USA. Correspondence to J.L.V. e‑mail: [email protected] doi:10.1038/nri3715 1

Despite many advances in our understanding of how the cells of the immune system react to beneficial antigens, as compared with those that are potentially harmful, there remain notable gaps in our knowledge of immune responses. In particular, there is still a strong need for a better understanding of how immune responses can be fine-tuned in order to elicit protective immunity to pathogens, to attenuate effector immune responses when they are no longer needed and to prevent the development of unwarranted immune responses that can lead to autoimmunity. The B7 molecules are members of the immuno­ globulin superfamily, and the study of these molecules at the end of the last millennium shed light on the complexity of the activatory and inhibitory co‑signals that T cells receive. The prototypical co‑stimulatory receptors that have garnered most attention are the antigenpresenting cell (APC)-expressed co‑stimulatory molecules B7.1 (also known as CD80) and B7.2 (also known as CD86), together with their T cell-expressed binding partners CD28 (which delivers an activatory signal to T cells) and cytotoxic T lymphocyte antigen 4 (CTLA4; which delivers an inhibitory signal to T cells). Over the years, these four founding members of the B7 family have been joined by many additional B7 superfamily members, including programmed cell death protein 1 (PD1), PD1 ligand 1 (PDL1), PDL2, B7RP1 (also known as ICOSL) and B7 homologue 3 (B7H3; also known as CD276). The recent discovery that another large family of immunoglobulin superfamily members — the butyro­ philins — may have a role in influencing and regulating immunity has provided us with a fresh opportunity to advance our knowledge of the molecular mechanisms involved in fine-tuning the immune response.

The first butyrophilin was described in the 1980s, when butyrophilin 1 (now known as butyrophilin subfamily 1 member A1 (BTN1A1)) was identified and shown to have a role in lactation — specifically, in the secretion, formation and stabilization of milk fat globules1,2 (BOX 1). Additional butyrophilin family members have since been identified, many of which are conserved between humans and mice (as reviewed in REFS 3–5) (TABLE 1). To date, the human butyrophilin family has been shown to have 13 members: BTN1A1, BTN2A1, BTN2A2, BTN2A3, BTN3A1, BTN3A2, BTN3A3, butyrophilin-like protein 2 (BTNL2), BTNL3, BTNL8, BTNL9, BTNL10 and SKINT-like (SKINTL). Given the complexity of this multigenic family, a revision to the orthologue nomenclature has recently been proposed4. The role of butyrophilins seems to stretch beyond the simple modulation of co‑stimulatory responses and extends to more influential functions in processes such as T cell selection, differentiation and cell fate determination. Genetic studies are revealing associations between polymorphisms in the genes encoding butyro­ philins and disease susceptibility in several different settings (BOX 2). This supports the notion that butyrophilins are important for immune homeostasis but we have a long way to go before we fully understand how these molecules — or the mutated forms of these molecules — interact with each other and with other immune modulators. Indeed, there are several butyrophilins for which no function has yet been identified (BOX 3). Once we have an improved understanding of butyrophilin activity at the molecular level, we may be in a better position to determine whether the targeting of butyrophilins might be beneficial in treating human disease.

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REVIEWS Box 1 | Butyrophilins and milk fat globule formation The molecule now known as butyrophilin subfamily 1 member A1 (BTN1A1) was the first butyrophilin to be identified and was discovered during investigation of milk fat globule (MFG) formation in the lactating mammary tissue of cows2. It is expressed on the apical surface of secretory cells in the mammary gland epithelium during the expulsion of newly synthesized milk2. Proteomic studies have since revealed that BTN1A1 is not the only butyrophilin that is present in MFGs52. Milk proteins can act as anti-inflammatory agents53. The MFG membrane is comprised of approximately 1–2% protein54,55. Butyrophilins are one of three major components of the human MFG membrane — the other two components being lactadherin (which can protect from rotavirus infection) and mucin 1 (MUC1; which can prevent Escherichia coli from binding to buccal epithelia)56. Whether butyrophilins have any anti-infective or anti-inflammatory activity, or simply provide scaffolding support, has long been debated. The non-redundant requirement for BTN1A1 in milk lipid secretion in mice during lactation was assumed to be owing to the binding of the B30.2 cytoplasmic domain of the butyrophilin to xanthine oxidoreductase, thus providing a scaffolding function that stabilizes the MFG membrane13,57. However, new studies have challenged the notion that the butyrophilins are present only for structural reasons. The presence of butyrophilins in networks of ridges in the residual plasma membrane of the MFG envelope, where it is tightly juxtaposed to butyrophilins in the monolayer derived from the secretory granule, suggests that MFG secretion might be controlled by interactions between butyrophilins at the plasma membrane and those that are in the secretory granule phospholipid monolayer58. The observed mobility of butyrophilins within the MFGs further indicates that interactions between butyrophilins and other proteins are very transient, and implicates a role for butyrophilins that extends beyond simple structural stability13.

In this Review, we focus on summarizing the unfolding biology of the various butyrophilin family members and we describe the evolving roles for these molecules in contributing to the maintenance of a healthy immune system. Early studies clearly revealed a role for butyrophilins in the regulation of co-stimulatory activities and the attenuation of immune reactivity in particular. More unexpectedly, perhaps, are the new insights into the regulation of biology beyond the inhibition of immune co-stimulation. As more research is directed at exploring this large family of molecules, it is quite possible that the range of biological functions will be extended even further.

The butyrophilin family Two lines of evidence initially guided research efforts towards exploring the immunological roles of butyro­ philins. First, the structural features of butyrophilins indicated that these proteins are very closely related, phylogenetically, to the B7 superfamily of co‑stimulatory molecules3,4,6,7. Second, many butyrophilins are expressed by immune cells and by cells that closely interact with immune cells, such as thymic stromal cells and intestinal epithelial cells (TABLE 1). Membership of the B7 family is mainly defined by the presence of extracellular IgV and IgC domains, a transmembrane region and a small intracellular cytoplasmic tail8 (FIG. 1). The IgV and IgC domains of B7 family members seem to be important for interacting with other immunoglobulin superfamily members. Many of the receptors and ligands of the B7 family share binding partners. For example, B7.1 and B7.2 both bind CD28 and CTLA4, and B7.1 has more recently been shown to interact with and bind to PDL1. Similar to the B7 family, members of the butyrophilin family typically have

two extracellular immunoglobulin domains and a trans­ membrane region, with the divergence between families relating to the fact that the butyrophilin and butyro­ philin-like family members also typically have a B30.2 intra­cellular signalling domain. The similarity of the butyrophilin IgV and IgC domain organization to that of B7 family members suggests that they may share binding mechanisms. However, as discussed below, there is still a massive void in our knowledge of the specific binding interactions and binding partners of the butyrophilins.

Binding partners and signalling mechanisms Cell-surface binding partners. BTN2A1 represents the first butyrophilin family member for which a cellsurface binding partner has been identified. BTN2A1 has been shown to bind to DC-specific ICAM3‑grabbing non-integrin (DC‑SIGN; also known as CD209), which is expressed by monocytes and dendritic cells 9. The importance of this biological interaction needs further study but there are suggestions that it could have a role in immune surveillance of tumours9. The binding of other butyrophilins to DC‑SIGN has not been reported, so it is uncertain whether this interaction is representative of some broad biology that is shared across family members or whether it is somewhat unique to BTN2A1. Although many of the studies that we discuss in this article demonstrate the ability of recombinant butyro­ philin molecules to bind to a variety of resting or activated immune cell types, the precise mechanism by which butyrophilins interact with other molecular entities on immune cells — and the nature of these molecules — remain to be identified. Assuming similarity to the B7 family, one might expect heterotypic interactions to occur in trans between butyrophilin molecules that are expressed on different cells. Studies seeking to better understand the binding interactions of the butyrophilins have mainly served to simply confirm that butyro­philins do not interact with B7 family members. Although they have been shown to interact with molecules that are expressed by T cells, BTN3A1 and BTNL8 do not seem to be able to bind CD28, CTLA4, inducible T cell costimulator (ICOS) or PD1 (REFS 10,11). Similarly, BTNL2 does not interact with any of a large panel of B7 family receptors and ligands that are expressed either on the cell surface or as soluble proteins12. The lack of information on the molecular determinants of the activities of the butyro­philins has hindered progress in exploiting these molecules as therapeutic targets. Intracellular binding interactions. Although the molecules of the butyrophilin and B7 families share a similar extracellular organization, the greatest divergence between these molecules is found within the intracellular domain. One structural feature that is worthy of mention is the B30.2 protein-binding domain (BOX 4) that is present in the intracellular tail of most butyrophilins, except for BTNL2 and BTN3A2. The B30.2 domain of BTN1A1 binds the enzyme xanthine oxidoreductase (XOR; also known as XDH) and this interaction stabilizes BTN1A1 within the membrane of the milk fat globule13,14. XOR does not

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REVIEWS Table 1 | The butyrophilin superfamily: cellular sources and interactions* Human gene

Mouse gene

Expression pattern

Interacting cell or molecular binding partner

BTN1A1

Btn1a1

Mammary glands, thymic stromal cells Activated T cells and macrophages and B cells

BTN2A1

–

Intestinal epithelial cells and broad immune cell expression

BTN2A2

Btn2a2

Dendritic cells, monocytes, B cells and Activated T cells thymic epithelial cells

BTN2A3

–

Unknown

Unknown

BTN3A1

–

Stressed cells, malignant cells and broad immune cell expression

B cells and T cells

BTN3A2

–

Malignant cells and broad immune cell expression

Unknown

5,10,88

BTN3A3

–

Broad immune cell expression

Unknown

5,10

–

Btnl1

Intestinal epithelial cells and macrophages

Activated T cells, B cells and dendritic cells

18,29

BTNL2

Btnl2

Intestinal epithelial cells, plasmacytoid dendritic cells and macrophages

T cells, B cells and vascular endothelium

5,12, 17,73

BTNL3

–

Neutrophils

Unknown

5

–

Btnl4

Intestinal epithelial cells

Unknown

29

–

Btnl5

Unknown

Unknown

–

Btnl6

Intestinal epithelial cells

Unknown

–

Btnl7

Unknown

Unknown

BTNL8

–

Neutrophils

Resting T cells

5,11

BTNL9

Btnl9

B cells

Activated T cells, B cells, dendritic cells and macrophages

5,18

BTNL10

Btnl10

Unknown

Unknown

SKINTL

–

Unknown

Unknown

–

Skint1

Thymic epithelial cells

Unknown

30

–

Skint2

Broad expression in lymphoid and non-lymphoid tissues

Activated T cells and antigen-presenting cells

19

–

Skint3–11

Unknown

Unknown

DC‑SIGN (expressed by dendritic cells and monocytes)

Refs 16,58 5,9 5,16,25

5,10,23,31

29

BTNxAy (where “x” and “y” are numbers), butyrophilin subfamily x member y; BTNL, gene encoding butyrophilin-like protein; DC‑SIGN, DC-specific ICAM3‑grabbing non-integrin; SKINTL, SKINT-like. “–” indicates that there is no known orthologue. *There are currently 13 genes that are known to encode members of the human butyrophilin superfamily. Although some family members show a high level of conservation across species, many differences are found between humans and mice (as extensively reviewed in REF. 4). Rather than exhaustively listing the tissues in which expression of the transcripts has been observed, this table catalogues the most compelling evidence of expression, with a focus on protein data.

seem to bind to the B30.2 domains of BTN2A1 or BTN3A1 (REF. 13) and it remains to be seen whether the B30.2 domain of other butyrophilins also binds to XOR. The intracellular B30.2 domain of BTN3A1 has, however, recently been reported to support the binding of prenylated phosphoantigens15. The binding of phosphoantigens to the intracellular B30.2 domain is reported to induce a conformational change in the extracellular domains of BTN3A1 and it has been proposed as a potential mechanism by which BTN3A1 on stressed cells triggers the activation of certain γδ T cells (as discussed in more detail below). The biological relevance of the B30.2 domain for immune signalling by butyrophilins other than BTN1A1 and BTN3A1 remains to be investigated.

In summary, there is still a substantial lack of knowledge on the intracellular and extracellular binding partners for butyrophilins, and on the mechanisms by which they signal to mediate their immunomodulatory functions. However, the use of recombinant butyrophilin–Fc fusion proteins has enabled the identification of the cells that interact with butyrophilins and some of the functional outcomes of these interactions, as we discuss below.

Regulation of T cell function by butyrophilins T  cell inhibition by butyrophilins. In vitro assays using recombinant human BTN1A1–Fc 16, human BTN2A2–Fc 16 , human BTNL2–Fc 12,17 and mouse BTNL1–Fc18 have shown that these butyrophilins can

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REVIEWS Box 2 | Genetic associations between butyrophilin polymorphisms and human disease Polymorphisms in BTNL2 (which encodes butyrophilin-like protein 2) were first reported to be associated with sporadic inclusion body myositis and sarcoidosis70,73,75–77. Multiple BTNL2 single nucleotide polymorphisms (SNPs) have now been identified that are associated with many different diseases46,63–73,75–81 (see the table). A large number of BTNL2 SNPs have been associated with inflammatory bowel disease but only five have been replicated between studies. These five SNPs are associated with susceptibility to ulcerative colitis but not Crohn’s disease69,80. The location of these SNPs within the exons encoding the membrane proximal IgC extracellular domain makes it intriguing to postulate that the polymorphisms might affect the ability of BTNL2 to dimerize (either with itself or with another B7 family member), although there are no functional data to support this hypothesis. As the BTNL2 gene is located in the MHC locus, some of the SNPs might be associated with the disease because they are in linkage disequilibrium with SNPs in the MHC, although there is some evidence showing that the linkage between BTNL2 SNPs and disease is independent of HLA genes73. In copy number variant (CNV) studies, a new gene-fusion product known as BTNL8*3 has been identified74. This CNV commonly occurs in Europeans, East Asians and Americans but is rarely found in African, Middle Eastern and Oceanic populations. The influence of the BTNL8*3 genotype on immunity is yet to be fully understood but, as with all CNV studies, there is likely to be some evolutionary selection pressure due to environmental influences. Individuals carrying the BTNL8*3 genotype exhibited differential (usually decreased) expression of a number of immunoregulatory genes, including those encoding cytokines, cytokine receptors, immune signalling molecules and matrix metalloproteinases74. The physiological importance of this novel BTNL8*3 protein in autoimmunity and inflammatory disease has yet to be uncovered but the expression data suggest that there may be potential implications for immunoregulatory control.

Gene

Association

BTN2A1

Dyslipidaemia, metabolic syndrome, myocardial infarction and chronic kidney disease

BTN3A2

Type 1 diabetes

38

BTN3A3

Ovarian cancer

45

BTNL2

Sarcoidosis, spontaneous inclusion body myositis, ulcerative colitis, Graves’ disease, type 1 diabetes, asthma (IgE responsiveness), systemic lupus erythematosus, rheumatoid arthritis and prostate cancer

BTNL8*3

Altered immunoregulatory gene expression

have an inhibitory effect on CD4+ T cell proliferation (FIG. 2a). In addition, human BTN1A1, BTN2A2 and BTNL2 and mouse BTNL1 were found to reduce T cell expression of many of the cytokines that are associated with T cell activation, including interleukin‑2 (IL‑2) and interferon‑γ (IFNγ)12,16–18. It is not only CD4+ T cell responses that can be inhibited by butyrophilins, as SKINT2 (also known as B7S3) has been found to inhibit CD8+ T cell activation in mice19. Box 3 | Butyrophilins with no known function Butyrophilin-like protein 9 (BTNL9) is expressed in a variety of tissues in humans and mice. Among isolated immune cell types, B cells show the strongest expression5. No known function as been ascribed to BTNL9 but recombinant BTNL9–Fc has been shown to bind to many immune cells including T cells, B cells, macrophages and dendritic cells18. With this profile of binding, it will not be surprising if BTNL9 joined the panel of butyrophilins with negative regulatory effects on immune cells. Despite the evidence for binding to immune cells, no functional modulation has been reported18. BTNL3 and BTN2A3 (which encodes butyrophilin subfamily 2 member A3) are found in humans only, and the Btnl4, Btnl5, Btnl6 and Btnl7 genes are found only in mice. To date, no functional activity has been associated with any of these molecules. The BTNL10 gene (also known as BUTR1) is found in humans and rodents. It is the newest molecule to be reported and thus it is hardly surprising that, as yet, nothing is known about its expression or function4. The SKINT subfamily of butyrophilins has a disproportionately large number of members in rodents (there are currently 11 in mice), whereas only a single orthologue exists in humans and seems to be a pseudogene (SKINTL; SKINT-like). As yet, there are no reported functions for SKINT subfamily members other than SKINT1 and SKINT2. The lack of ascribed functions for these butyrophilins is most probably the result of inadequate study of the molecules, rather than owing to a true lack of biological importance.

Refs 39–43,59–62

46,63–73, 75–81 74

Butyrophilins have also been shown to regulate immune responses in vivo. Treatment with recombinant BTN1A1 inhibited T cell activation and protected against disease development in animal models of experimental autoimmune encephalomyelitis (EAE)20,21. These findings have been interpreted to be related to the induction of bystander tolerance owing to the similarity of BTN1A1 to myelin oligodendrocyte glycoprotein (MOG) but it is interesting to speculate that BTN1A1 may perhaps have mediated inhibition more directly by modulating T cell activation20,21. Conversely, treatment with neutralizing antibodies specific for a different family member, BTNL1, resulted in T cell activation and exacerbated effector T cell responses in animal models of immunization, autoimmunity and allergy 18, suggesting that blockade of the inhibitory effect of butyro­ philins can unleash the immune response. Further studies should help to clarify whether the butyrophilin family members have redundant or non-redundant roles in these disease settings, and the use of mice that are deficient in different butyrophilins should be particularly helpful in this regard. The BTN3A subfamily of butyrophilins (BTN3A1, BTN3A2 and BTN3A3) are expressed by most human immune cell subsets, including T cells, B cells, monocytes, dendritic cells and natural killer (NK) cells. This broad expression pattern has made this group of butyrophilins among the best studied10,22,23. A number of antibodies that are specific for BTN3A molecules (also known as CD277) have been reported

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REVIEWS B7 family

IgV

IgC

TM

Butyrophilin family

IgV

IgC

TM

IgV

IgC

TM

BTNL3

IgV

IgC

TM

B30.2

BTNL8

IgV

IgC

TM

B30.2

BTNL9

IgV

IgC

TM

B30.2

BTNL10

IgV

IgC

TM

B30.2

BTNL2

Butyrophilinlike family

IgV

IgC

B30.2

Figure 1 | Structural comparison of the B7 and butyrophilin superfamilies.  Nature Reviews | Immunology The hallmark feature of the B7, butyrophilin and butyrophilin-like (BTNL) families is the inclusion of extracellular immunoglobulin domains, typically an IgV and an IgC domain. In some cases, like BTNL2 and B7 homologue 3 (B7H3), these domains are repeated resulting in four extracellular immunoglobulin domains. No B7 family members that have been identified to date contain recognizable intracellular signalling domains, whereas this is a common feature of the butyrophilin and BTNL families, with only butyrophilin subfamily 3 member A2 (BTN3A2) and BTNL2 lacking this feature. The second immunoglobulin domains of BTNL3, 8, 9 and 10 are less well characterized. TM, transmembrane domain.

in the literature, although none of these seem able to distinguish between the three BTN3A family members10,23. Despite this, the biological impact of the different antibodies is quite different. In in vitro studies, the BTN3A‑specific monoclonal antibody 232.5 was found to bind to BTN3A directly on the T cell surface, to drive the phosphorylation of the subfamily member BTN3A3, and to initiate an inhibitory signalling cascade that resulted in decreased T cell proliferation and cytokine production23 (FIG. 3b). This effect required BTN3A aggregation and was mediated independently of regulatory T (TReg) cell function 23. Studies using an alternative BTN3A‑specific antibody (103.2) confirmed the observation that BTN3A‑specific antibodies can inhibit T cell activation but this was demonstrated to be via a completely different mechanism24. Rather than mediating its effect via BTN3A3, like the agonistic BTN3A‑specific antibody 232.5, the BTN3A‑specific antibody 103.2 seems to behave as an antagonist and to mediate its effect by modulation of BTN3A1. Antibody 103.2 seems to be able to bind BTN3A1 either monovalently or bivalently, and it is assumed to mediate its inhibitory effect by sterically preventing BTN3A1 from engaging with cellular activation machinery 24 (FIG. 3b). Activatory co-stimulation of T cells by butyrophilins. Although most of the data suggest that butyrophilins attenuate or inhibit immune cell activation, there are now some data showing that butyrophilins can also augment T cell activation. In contrast to most other butyro­ philin molecules, which mainly bind to activated cell types, studies using BTNL8–Fc suggest that BTNL8 primarily binds to resting T cells11. BTNL8 is expressed in humans, but not in rodents, and its expression is particularly high in neutrophils5,11. The addition of BTNL8–Fc to T cell cultures enhanced proliferation in vitro (FIG. 2b)

and this was associated with the increased production of both T helper 1 (TH1)‑type and TH2‑type effector cytokines — including IFNγ and tumour necrosis factor (TNF) — and with the increased production of regulatory cytokines, such as IL‑10 (REF. 11). The in vivo administration of BTNL8–Fc to mice enhanced antigenspecific primary (but not secondary) antibody responses associated with immunization11. It is not yet clear where BTNL8 ranks in terms of its importance relative to B7.1 and B7.2 in modulating naive or resting T cell reactivity and further study will be needed to determine whether there is any unique role for this molecule. Unlike the BTN3A‑specific monoclonal antibodies 232.5 and 103.2 that are described above, the addition of the BTN3A‑specific monoclonal antibody 20.1 to T cell cultures increased proliferation and cytokine production by agonizing T cells10,22,24 (FIG. 3c). The agonistic 20.1 antibody is reported to bind to BTN3A more weakly than antibody 103.2 but it is not the binding affinity per se that is thought to drive the opposing biological outcomes. Rather, BTN3A‑specific antibody 20.1 seems to bind to a different epitope on BTN3A compared with antibody 103.2, which results in crosslinking of the BTN3A molecules on the cell surface and triggering of cellular activation24. More information is needed to fully understand why different butyrophilin antibodies have agonistic or antagonistic properties, particularly regarding whether the stoichiometry of antibody binding is consistently the sole driver of differential activity in all cells and in all culture conditions. TReg cell induction by butyrophilins. Within the past year, two groups have reported the ability of butyro­ philins to induce the differentiation of naive CD4 + T cells into TReg cells25,26 (FIG. 2c). In vitro experiments using different combinations of co‑stimulatory molecules have shown that BTNL2 administered with either B7.1 or B7.2 can induce the development of forkhead box P3 (FOXP3)-expressing T cells with a regulatory cell phenotype and function26. BTNL2 seems to mediate this effect by blocking the AKT-mediated inactivation of the transcription factor forkhead box protein O1 (FOXO1), which is necessary for FOXP3 expression and TReg cell differentiation. The BTNL2‑induced TReg cells that were differentiated in this manner in vitro seem to be most similar to thymus-derived (also known as ‘naturally occurring’) TReg cells. The BTNL2‑dependent TReg cells that were induced in vitro maintained their regulatory activity in vivo and they were able to attenuate intestinal inflammation in a mouse model of colitis26. Notably, BTNL2 is expressed at the highest levels in the intestine, a site in which TReg cell activity is instrumental for maintaining immune homeostasis in the presence of constant antigenic onslaught. BTN2A2 can also alter T cell receptor (TCR)mediated activation and induce de  novo FOXP3 expression in T cells via the inhibition of AKT phosphorylation and the inhibition of extracellular signalregulated kinase 1 (ERK1) and ERK2 (REF. 25). Similar to BTNL2‑induced FOXP3+ cells, BTN2A2‑induced FOXP3 + cells also exhibited regulatory properties.

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REVIEWS Box 4 | The B30.2 domain A typical, though not universal, feature of the butyrophilin family is the presence of a large intracellular domain known as the B30.2 domain. The B30.2 domain is defined by the presence of three specific sequence motifs (LDP, WEVE and LDYE) and is also known as a PRY/SPRY domain owing to its inclusion of a SPRY domain preceded by an amino‑terminal PRY motif82,83. The B30.2 domain is not exclusive to the butyrophilin family, as it has been identified in 51 genes in the human genome, with an additional 52 genes containing the SPRY domain without N‑terminal PRY84. These domains seem to be important in mediating protein–protein interactions, though in many cases the binding partners have not been identified. Although B30.2 domain-containing proteins have diverse roles, many have been reported to be important in immune function, including the TRIM genes (which encode tripartite motif proteins) that are located in the MHC cluster and pyrin (as reviewed in REF. 85). For example, the pyrin B30.2 domain is required for the cleavage of pyrin — which can lead to the inhibition of the processing of interleukin-1β (IL‑1β) — and mutations in this region can cause familial Mediterranean fever, an autosomal recessive inflammatory disorder86,87. Within the butyrophilin family, B30.2 domain function has been demonstrated for butyrophilin subfamily 1 member A1 (BTN1A1) and BTN3A1. The B30.2 domain of BTN1A1 reportedly binds to xanthine oxidoreductase (XOR) to participate in milk fat globule secretion13. The role of this interaction in immune cells is unclear but it is hypothesized that the BTN1A1–XOR interaction may lead to the production of reactive oxygen species, which have immunomodulatory and antimicrobial functions. More recently, structural and functional studies have demonstrated that the B30.2 domain of BTN3A1 is capable of binding to prenyl pyrophosphates and that this causes a conformational change in the extracellular domain of BTN3A1, which then leads to the activation of a specialized subset of γδ T cells15.

BTN2A2 is expressed at high levels on thymic epithelial cells, as well as on conventional and non-conventional APCs, such as dendritic cells, monocytes and B cells. Whether the main function of BTN2A2 is to facilitate the selection of TReg cells in the thymus or whether it is to promote the differentiation of TReg cells in the periphery remains to be determined (FIG. 2c).

Regulation of γδ T cells by butyrophilins γδ T cell selection, differentiation and fate. In mice, Skint1 mRNA is expressed in the thymus and skin, which are both sites at which extensive interaction occurs between stromal cells and T cells, and these interactions are often associated with γδ T cell selection and function. Studies in mice bearing a mutation in Skint1 (FVBTac mice from Taconic) demonstrated that SKINT1 expression in thymic epithelial and stromal cells is required for the positive selection of a specialized population of γδ T cells in the thymus (FIG. 2d) — namely, the Vγ5 +Vδ1 + T cells that traffic to epidermal sites, such as the skin, upon exiting the thymus 27 (FIG. 2e) . Further interrogation of the thymic selection process has revealed that SKINT1 drives the differentiation of fetal thymocytes towards an IFNγ + phenotype in an early growth response protein 3 (EGR3)‑dependent manner 28. The differentiation of fetal thymocytes into IFNγ-producing Vγ5+Vδ1+ T cells comes at the expense and exclusion of the retinoic acid receptor-related orphan receptor-γt (RORγt)-dependent Vγ6 +Vδ1+ T cells that produce copious quantities of IL‑17. These studies suggest that the functional specialization of γδ T cells occurs early during development and that it is influenced

by butyrophilins. Although it has been difficult to verify substantial expression of SKINT1 on the cell surface, its expression can be induced30 and its influence on γδ T cell biology seems to be consistent with a role in thymic selection. Unexpectedly, the deficit in the positive selection of Vγ5+Vδ1+ T cells that occurs in the thymus of the Skint1‑mutant FVBTac mice is not associated with an absolute decrease in γδ T cell numbers in the epidermis of the skin — only the canonical Vγ5+Vδ1+ T cells are missing. The authors have interpreted this observation to mean that SKINT1 expression in keratinocytes may be required for retaining Vγ5+Vδ1+ cells at epidermal sites30 (FIG. 2e). The relevance of these findings to γδ T cell biology in human skin is yet to be fully understood. Cellular stress and antigen presentation. The most recent data to unfold regarding butyrophilin biology suggest that BTN3A1 may affect antigen recognition by, and antigen presentation to, Vγ9+Vδ2+ T cells in humans. Vγ9+Vδ2+ T cells are thought to be the first line of defence during infection. They have long been known to respond to phosphorylated prenyl meta­ bolites, which can be derived from the host or from microorganisms, and they accumulate in meta­bolically distressed cells. Despite data demonstrating that the Vγ9+Vδ2+ T cells can specifically recognize these phosphorylated prenyl non-peptide phospho­antigens as conserved ‘danger-associated’ epitopes, it has been historically difficult to show a direct interaction between these antigens and the Vγ9Vδ2 TCR. In initial studies, the binding of agonistic BTN3A‑specific monoclonal antibody 20.1 to Vγ9+Vδ2+ T cells mimicked how these cells responded to the phosphoantigens and antagonistic BTN3A‑specific monoclonal antibody 103.2 inhibited the activity 24,31–33. Through domain shuffling and small interfering RNA (siRNA) studies, BTN3A1 was identified as the BTN3A family member that was able to confer this effect 31,32. The differential activity of these two BTN3A‑specific monoclonal antibodies (which both bind to all three BTN3A proteins) was originally thought to arise due to the oligomerization of BTN3A molecules on the T cell surface, thereby altering the availability of the intracellular B30.2 domain either to bind to co-signalling moieties24 or to sense prenyl pyrophosphates31. The most recent study designed to more thoroughly characterize the structure and function of the intracellular B30.2 domain of BTN3A1 has revealed the presence of a positively charged pocket that is capable of binding intracellular phosphoantigens15 (FIG. 3a). Elegant mutagenesis studies have demonstrated that changing a single amino acid in this pocket is sufficient to confer the ability to bind intracellular phospho­antigens to the B30.2 domain of another family member, BTN3A3 (REF. 15). The binding of phosphoantigens to the intracellular B30.2 domain of BTN3A1 seems to alter the conformation of extracellular BTN3A1 and it is this alteration that drives the activation of Vγ9+Vδ2+ T cells15.

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REVIEWS a Resting or activated T cell

b

Resting T cell

Treatment with: • BTN1A1–Fc • BTN2A2–Fc • BTNL2–Fc • BTNL1–Fc

T cell inhibition • Decreased proliferation • Decreased cytokine production

Treatment with BTNL8–Fc

T cell activation • Increased proliferation • Increased cytokine production

c

Peripherally induced TReg cell

Intestinal epithelial cell BTNL2 Thymic epithelial cell Monocyte B cell

TReg cell differentiation • Inhibition of AKT and ERK • Induction of FOXO1 • Induction of FOXP3 • Immunosuppressive functions

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Thymus-derived or peripherally induced TReg cell

d

e

IFNγ

SKINT1

Thymic epithelial or stromal cell

γδ T cell development Immature thymocyte

Vγ5Vδ1 T cell

Keratinocyte γδ T cell trafficking or retention SKINT1

Vγ5Vδ1 T cell

Figure 2 | Modulation of T cell activity by butyrophilins.  Butyrophilins have| Immunology been shown Nature Reviews to modulate T cell activation, to induce regulatory T (TReg) cell differentiation, to mediate T cell selection in the thymus and to promote T cell trafficking and retention. a | T cell activation can be inhibited by co‑culture with butyrophilin subfamily 1 member A1 (BTN1A1), BTN2A2, butyrophilin-like protein 1 (BTNL1) or BTNL2 resulting in attenuated proliferation and cytokine production. b | The activation of resting T cells can be enhanced by co‑culture with BTNL8, which results in increased proliferation and cytokine production. c | BTNL2 and BTN2A2 can induce TReg cell differentiation. BTNL2 seems to induce the differentiation of TReg cells from mature naive CD4+ T cells. BTN2A2 may be involved in thymic selection of TReg cells, as well as the differentiation of TReg cells from mature naive CD4+ T cells. d | The expression of SKINT1 on thymic epithelial cells has been demonstrated to mediate the positive selection of Vγ5Vδ1 T cells. e | Keratinocyte expression of SKINT1 drives Vγ5Vδ1 trafficking and/or retention in the skin. ERK, extracellular signal-regulated kinase; IFNγ, interferon-γ; FOXO1, forkhead box protein O1; FOXP3, forkhead box P3.

An alternative model has also been put forward in which the distal IgV domain of BTN3A1 has been proposed to form a shallow pocket permitting phosphorylated prenyl antigens to bind extracellularly, conferring the ability of BTN3A1 to behave like a classical MHC class I or MHC class II molecule by presenting phospho­ antigens on the cell surface to Vγ9 +Vδ2+ T cells34–36 (FIG. 3a). Clearly the biology is complicated and there is still substantial discussion in the field regarding which models are correct.

Effects of butyrophilins on innate immune cells Research efforts have mainly focused on the effects of the butyrophilin family on T cell responses but data are slowly emerging for their role in other cell types. Recent data have shown that antibody-mediated activation of BTN3A can drive inflammatory signalling events in cell types other than T cells. Monocytes and monocyte-derived dendritic cells both express BTN3A1, and the addition of the BTN3A‑specific monoclonal antibody 19.5 to cultures of these cells was shown to drive increased cell survival and to prevent apoptosis37 (FIG. 3c). The addition of the antibody to these cultures also enhanced co-stimulatory molecule expression by monocytes and dendritic cells, and increased IL‑1, IL‑8 and IL‑12 production by both cell types37. The activation of monocytes and monocytederived dendritic cells by BTN3A‑specific antibodies could be further increased by Toll-like receptor (TLR) signalling, perhaps suggesting that the major role of the BTN3A subfamily is to enhance and amplify pro-inflammatory signals that are initiated by other receptors37. BTN3A molecules are also expressed by NK cells and there is evidence to suggest that BTN3A1 and BTN3A2 differentially regulate the production of cytokines, such as IFNγ22. In experiments using transfected cells and the BTN3A‑specific monoclonal antibody 20.1, the engagement of BTN3A2 (the BTN3A member that is predominantly expressed by NK cells) was shown to decrease IFNγ production downstream of NKp30 (also known as NCR) signalling, whereas the engagement of BTN3A1 was found to increase NKp30‑induced IFNγ production22. These data suggest that the different BTN3A isoforms may have opposing roles in regulating NK cell function. Epithelial cell and immune cell crosstalk Intestinal epithelial cells and the cells of the mucosal immune system are in extremely close proximity, which facilitates crosstalk between these populations. BTNL1, a family member that is found in mice but not in humans, is expressed by epithelial cells that line the small intestine29. The recent description of how BTNL1 on epithelial cells can modulate the epithelial response to intra­epithelial lymphocyte (IEL) signals sheds light on a new tier of interplay that may have consequences for how protective immunity is controlled at mucosal surfaces29. Epithelial cell expression of BTNL1 attenuates the ability of these cells to produce pro-inflammatory cytokines and chemokines, such as IL‑6, IL‑15, CXC-chemokine ligand 1 (CXCL1) and CC-chemokine ligand 4 (CCL4), in response to signals that are delivered by activated IELs. Whether the primary rationale for this type of biology is to keep the intestinal environment in a controlled state of inflammation or whether it is to prevent hyper-reactivity to beneficial antigens — such as those from food and the commensal flora — remains to be determined. Intestinal epithelial cells are able to present antigens to IELs and thus probably have a very strong influence on the reactivity of this unusual lymphocyte population. The high expression of BTNL2 in intestinal epithelial cells and the ability of BTNL2 to attenuate T cell responsiveness support the notion that the epithelium may have a role in promoting tolerance or intestinal homeostasis12.

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REVIEWS a

Phosphoantigen BTN3A1

Vγ9Vδ2 TCR

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Stressed APC

b

BTN3A-specific antibody (e.g. 232.5 or 103.2)

T cell BTN3A

c

Antigen presentation • Prenyl pyrophosphoantigens have been proposed to bind either in a pocket in the intracellular B30.2 domain or in a shallow groove in the extracellular immunoglobulin domain of BTN3A1 • Phosphoantigen binding allows BTN3A1 to specifically activate Vγ9+Vδ2+ T cells

T cell inhibition • Agonistic 232.5 antibody phosphorylates BTN3A3 and initiates inhibitory signalling • Antagonistic 103.2 antibody sterically blocks association of proteins engaged by BTN3A1 during activation

BTN3A-specific antibody (e.g. 20.1 or 19.5) Cell activation • Agonistic 20.1 or 19.5 antibody delivers activatory signal to T cells, monocytes and dendritic cells • Enhanced proliferation, survival and cytokine production

T cell

Monocyte Dendritic cell

d Immune evasion Expression of BTN3A by tumour cells attenuates antitumour T cell responses

T cell Tumour

e

BTN3A-specific antibody Tumour immunotherapy Blocking antibodies specific for BTN3A permit antitumour T cell responses

T cell

Figure 3 | The many functions of the butyrophilin 3A subfamily. Members of the Nature Reviews | Immunology butyrophilin 3A (BTN3A) subfamily have many functions, the nature of which seems to depend on the model system and reagents that are used. These functions include the modulation of T cell function, antigen presentation and immune evasion. BTN3A‑specific antibodies can transmit either inhibitory or activating signals depending on the reagent that is used. a | BTN3A1 can bind to prenyl pyrophosphate antigens that are derived from microorganisms and/or the host, and drive the activation of human Vγ9+Vδ2+ T cells, although the precise binding site of phosphoantigens is controversial. b | BTN3A‑specific monoclonal antibodies such as 232.5 or 103.3 directly bind to T cells and transmit inhibitory signals. c | BTN3A‑specific monoclonal antibodies such as 20.1 or 19.5 directly bind to T cells or other immune cells and transmit activatory signals. d | The role of BTN3A in tumour immunity is complicated and not well understood. The expression of BTN3A in the tumour microenvironment may contribute to immune evasion by attenuating T cell activity. e | Blockade of BTN3A expression on malignant cells may release T cell constraints that are imposed by BTN3A engagement and thus elicit antitumour immunity. APC, antigen-presenting cell; TCR, T cell receptor.

The reported prominence of BTNL4 and BTNL6 in mouse epithelial cells also suggests a noteworthy role for butyrophilin biology in epithelial cell and immune cell crosstalk in the gut, although the precise context is yet to be established12,29.

Butyrophilins and disease Butyrophilins as genetic modifiers of disease susceptibility. The majority of inflammatory diseases are thought to be polygenic in nature and to arise as a result of a combination of environmental factors and genetic pre­ disposition. Mutations in butyrophilin genes (BOX 2) are not likely to be the only factors contributing to disease susceptibility. It is possible, though, that these polymorphisms may be more influential in altering disease outcome, rather than actually predisposing to disease, and thus they may be useful for stratifying patients for therapy. Supporting this hypothesis, polymorphisms in BTN3A2 are associated with susceptibility to type 1 diabetes and this has been replicated in two cohorts38. Unexpectedly, BTN2A1 may represent a susceptibility gene for metabolic syndrome and myocardial infarction through an effect on dyslipidaemia39–43, as individuals with even one copy of the less frequent allele were more likely to have elevated serum cholesterol, lowdensity lipoprotein (LDL) and triglycerides41, although the mechanistic basis for these effects on metabolism remains to be elucidated. Butyrophilins and immune evasion by cancer cells. High levels of expression of the BTN3A subfamily of butyro­ philins on either malignant or stromal cells in ovarian and hepatocellular cancer has been hypothesized to contribute to immune evasion by attenuating the activity of tumour-infiltrating T cells44 (FIG. 3d). The upregulation of inhibitory butyrophilins by soluble mediators, such as CCL3 or vascular endothelial growth factor (VEGF) — which are highly expressed in the tumour micro­ environment — probably also contributes to decreased immune surveillance of the tumour 44. However, our understanding of the roles of butyrophilins in anti­ tumour immunity is still at a preliminary stage and further experiments will be needed to clarify their functions in the tumour microenvironment. Further supporting the notion that butyrophilins might contribute to immune evasion in oncology settings, single nucleotide polymorphisms (SNPs) in BTN3A3 and BTNL2 have recently been reported to be associated with increased susceptibility to ovarian45 and prostate cancer 46, respectively. The functional modification resulting from these SNPs is not fully understood but the upregulation of inhibitory butyrophilins in the tumour microenvironment might dampen antitumour immunity.

Therapeutic potential of targeting butyrophilins The past two decades have seen dramatic advances in the development of new therapies. For example, the biological TNF inhibitors and small-molecule Janus kinase (JAK) inhibitors have dramatically improved the lives of many patients with chronic inflammatory diseases, such as rheumatoid arthritis and psoriasis47. Furthermore, the exciting results from recent studies using PD1‑specific immunotherapy for cancer suggest that this approach holds substantial promise48. Despite these advances, there are still large subsets of patients for whom the currently available therapies are ineffective or

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REVIEWS not tolerated, and it is for this reason that future efforts to identify novel, targeted and personalized approaches to therapy are warranted. Whether the need is to tame an overactive immune response in the context of inflammatory disease or to kick-start an immune response to eliminate cancer, the specificity offered by personalized immunotherapy is highly sought. Perhaps it is time for immunotherapy with butyrophilins to be gaining traction? Preclinical data suggest that the modulation of the immune response by recombinant butyrophilins has some benefit in animal models of disease11,20,21 but the translational potential of these observations is yet to be determined in human studies. Taming T cells. The overarching biological effects that are driven by many butyrophilins in vitro dampen and control immune reactivity. It is therefore extremely tempting to speculate that recombinant molecules might provide therapeutic benefit for patients with an over­active immune response, such as that seen in auto­immune and inflammatory diseases. Unfortunately, there is very limited data to demonstrate that the administration of recombinant forms of butyrophilins can either prevent the initiation of, or overcome existing, inflammatory responses in animal models of disease. It is not known whether this is because the recombinant inhibitory molecules have not been tested in many animal models of disease, whether it reflects the inability of the recombinant molecules to conform to a structurally relevant configuration, whether it reflects an inability of the recombinant molecule to circulate to the site of inflammation, or whether it simply means that butyrophilins have no role in modulating inflammatory processes. Although it is tempting to postulate, definitive studies will be needed before there is a clear path forward. In addition to the ability of recombinant butyrophilins to promote negative or inhibitory signals, the observation that butyrophilins such as BTNL2 or BTN2A2 can have a role in driving the differentiation and expansion of cells with a regulatory phenotype is an attractive therapeutic strategy. If it were possible to devise a therapy whereby host immune cells could be differentiated and converted to have a regulatory cell function, it would have enormous potential. Whether the expansion of TReg cells is sufficient to dampen an immune response is not clear, and whether it would be necessary to sustain the constant differentiation of cells into a regulatory phenotype would need to be determined. It seems that we are still many years from demonstrating the therapeutic benefit that may be achieved by enhancing TReg cell numbers and function, and whether there is any role for butyrophilins in this process is pure speculation at this moment in time. For butyrophilins (such as the BTN3A subfamily) that are expressed on T cells, it has been shown in vitro that agonistic antibodies can mediate an inhibitory signal that decreases T cell activation and proliferation. Immunomodulatory therapy via agonistic antibodies to the inhibitory butyrophilins that are expressed on T cells might offer an alternative novel therapeutic approach for treating autoimmune disease23. The selection of an antibody with the desired inhibitory function on T cells

will be incredibly important, as contrary data also exist to suggest that different BTN3A‑specific antibodies can have opposing effects on T cell activation10,22,24. Furthermore, it is also possible that the inhibition of T cell activation that is mediated by BTN3A expressed on non‑T cells might be inadvertently reversed by treatment with BTN3A‑specific monoclonal antibodies49. Given the contradictory and opposing functional consequences that different BTN3A‑specific antibodies have been demonstrated to elicit in T cells, considerable care would need to be taken before any targeted therapies should be considered and even then, extensive pre­ clinical testing will undoubtedly be necessary to ensure that the desired functional outcome is achieved safely. Immunotherapy for enhancing immunity. Given the promising clinical outcomes that have been observed with PD1‑specific therapy in cancer, it is timely to speculate that the butyrophilin pathway might hold promise for therapeutic intervention. The observation that butyro­ philins such as BTN3A1 are expressed in the tumour microenvironment and seem to dampen antitumour immunity makes therapy with blocking or neutralizing BTN3A‑specific antibodies an attractive therapeutic approach49 (FIG. 3e). However, the very recent discoveries implicating a role for BTN3A1 in triggering antigen recognition by γδ T cells in situations of cellular stress has led to a completely different hypothesis — that is, antitumour immunotherapy approaches that are based on Vγ9Vδ2 TCR agonists, such as synthetic phosphorylated agonists50,51, may be enhanced if agonistic BTN3A‑specific antibodies are used in combination31. Further exploration of these combination therapies will obviously be needed before advancing into human clinical trials, especially since opposing and/or reciprocal mechanisms for either blocking or activating BTN3A have been demonstrated to be beneficial in different experimental settings depending on the cell that is being targeted.

Conclusions So, where are we? The past decade has opened the door to a new — and incompletely understood — area of biology stemming from a family of molecules that had no previously ascribed immune function. The family is large and although there is some phylogenetic divergence, there are a substantial number of family members that seem to be conserved across species — both in terms of their expression and observed function. The biggest advances have come from the observation that many of the butyrophilins have the ability to modulate immune reactivity, either alone or in combination with B7 family members. That some butyrophilins can attenuate or enhance T cell activation adds a layer of complexity to our understanding of co-stimulation, in both health and disease. The additional observations that butyrophilins can alter T cell differentiation and T cell fate open the door to further studies that will be necessary to take full advantage of these properties. Furthermore, the functions of some butyrophilins remain unknown and further investigation of these may offer a ripe opportunity for more novel science to emerge.

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REVIEWS We still lack a comprehensive and thorough understanding of butyrophilin expression patterns and binding interactions. It is important to bear in mind that information on butyrophilin gene expression is more readily obtainable than information on protein expression levels, and there has not yet been any systematic attempt to document expression or binding patterns for all of the butyrophilin family members using a single platform. Thus, the data that have been described to date probably reflect the biases of ongoing research interests of certain laboratories, rather than meaningful differences between the family members. A detailed study of cellular and tissue distribution is now warranted, as is an unbiased search for the counter­structures which bind to butyrophilins. It will be crucial to identify these molecular interactions if there is to be any hope of developing future immuno­ therapies that target butyrophilins, whether this is inhibitory therapy for treating an overactive immune response in autoimmune and inflammatory disease or stimulatory therapy for enhancing immune responses to fight cancer. When we have a better understanding

Heid, H. W., Winter, S., Bruder, G., Keenan, T. W. & Jarasch, E. D. Butyrophilin, an apical plasma membrane-associated glycoprotein characteristic of lactating mammary glands of diverse species. Biochim. Biophys. Acta 728, 228–238 (1983). 2. Franke, W. W. et al. Antibodies to the major insoluble milk fat globule membrane-associated protein: specific location in apical regions of lactating epithelial cells. J. Cell Biol. 89, 485–494 (1981). 3. Abeler-Dorner, L., Swamy, M., Williams, G., Hayday, A. C. & Bas, A. Butyrophilins: an emerging family of immune regulators. Trends Immunol. 33, 34–41 (2012). This review provides a good summary of butyrophilin expression patterns. 4. Afrache, H., Gouret, P., Ainouche, S., Pontarotti, P. & Olive, D. The butyrophilin (BTN) gene family: from milk fat to the regulation of the immune response. Immunogenetics 64, 781–794 (2012). This paper provides a comprehensive phylogenetic analysis of the relationships between mammalian butyrophilin family members and a proposal for a new naming convention. 5. Arnett, H. A., Escobar, S. S. & Viney, J. L. Regulation of costimulation in the era of butyrophilins. Cytokine 46, 370–375 (2009). 6. Tazi-Ahnini, R. et al. Cloning, localization, and structure of new members of the butyrophilin gene family in the juxta-telomeric region of the major histocompatibility complex. Immunogenetics 47, 55–63 (1997). 7. Rhodes, D. A., Stammers, M., Malcherek, G., Beck, S. & Trowsdale, J. The cluster of BTN genes in the extended major histocompatibility complex. Genomics 71, 351–362 (2001). 8. Ikemizu, S. et al. Structure and dimerization of a soluble form of B7‑1. Immunity 12, 51–60 (2000). 9. Malcherek, G. et al. The B7 homolog butyrophilin BTN2A1 is a novel ligand for DC‑SIGN. J. Immunol. 179, 3804–3811 (2007). 10. Compte, E., Pontarotti, P., Collette, Y., Lopez, M. & Olive, D. Frontline: characterization of BT3 molecules belonging to the B7 family expressed on immune cells. Eur. J. Immunol. 34, 2089–2099 (2004). 11. Chapoval, A. I. et al. BTNL8, a butyrophilin-like molecule that costimulates the primary immune response. Mol. Immunol. 56, 819–828 (2013). This is one of the few papers demonstrating that recombinant butyrophilins can activate and enhance T cell responses. 12. Arnett, H. A. et al. BTNL2, a butyrophilin/B7‑like molecule, is a negative costimulatory molecule modulated in intestinal inflammation. J. Immunol. 178, 1523–1533 (2007). 1.

of how, and to what, butyrophilins bind, we should be able to better interrogate and dissect the intracellular signalling pathways. The ongoing genome-wide genetic studies continue to identify patient subsets with polymorphisms in the genes that encode butyrophilins. As the field moves towards full genome sequencing, it is possible that we may identify families carrying rare, but penetrant, mutations in one or more butyrophilins. Such discoveries will be useful for accelerating our understanding of the relative importance of butyrophilins in host immunity and health. In conclusion, it is apparent that we are still missing some vital pieces of information regarding butyrophilin biology. The next decade should be a fruitful time for addressing some of these major gaps in our understanding but it will require a diligent and dedicated approach. The technologies for evaluating expression already exist and the techniques for identifying binding partners should be sufficiently evolved to be of benefit to this endeavour. When these gaps in our understanding are reduced, it is possible that we may see the evolution of butyrophilin-targeted therapies.

13. Jeong, J. et al. The PRY/SPRY/B30.2 domain of butyrophilin 1A1 (BTN1A1) binds to xanthine oxidoreductase: implications for the function of BTN1A1 in the mammary gland and other tissues. J. Biol. Chem. 284, 22444–22456 (2009). 14. Vorbach, C., Scriven, A. & Capecchi, M. R. The housekeeping gene xanthine oxidoreductase is necessary for milk fat droplet enveloping and secretion: gene sharing in the lactating mammary gland. Genes Dev. 16, 3223–3235 (2002). 15. Sandstrom, A. et al. The intracellular B30.2 domain of butyrophilin 3A1 binds phosphoantigens to mediate activation of human Vγ9Vδ2 T cells. Immunity 40, 490–500 (2014). This paper demonstrates that the intracellular B30.2 domain of butyrophilins can mediate a biological effect that results in the activation of γδ T cells. 16. Smith, I. A. et al. BTN1A1, the mammary gland butyrophilin, and BTN2A2 are both inhibitors of T cell activation. J. Immunol. 184, 3514–3525 (2010). 17. Nguyen, T., Liu, X. K., Zhang, Y. & Dong, C. BTNL2, a butyrophilin-like molecule that functions to inhibit T cell activation. J. Immunol. 176, 7354–7360 (2006). 18. Yamazaki, T. et al. A butyrophilin family member critically inhibits T cell activation. J. Immunol. 185, 5907–5914 (2010). 19. Yang, Y. et al. Characterization of B7S3 as a novel negative regulator of T cells. J. Immunol. 178, 3661–3667 (2007). 20. Mana, P. et al. Tolerance induction by molecular mimicry: prevention and suppression of experimental autoimmune encephalomyelitis with the milk protein butyrophilin. Int. Immunol. 16, 489–499 (2004). 21. Stefferl, A. et al. Butyrophilin, a milk protein, modulates the encephalitogenic T cell response to myelin oligodendrocyte glycoprotein in experimental autoimmune encephalomyelitis. J. Immunol. 165, 2859–2865 (2000). 22. Messal, N. et al. Differential role for CD277 as a co‑regulator of the immune signal in T and NK cells. Eur. J. Immunol. 41, 3443–3454 (2011). 23. Yamashiro, H., Yoshizaki, S., Tadaki, T., Egawa, K. & Seo, N. Stimulation of human butyrophilin 3 molecules results in negative regulation of cellular immunity. J. Leukoc. Biol. 88, 757–767 (2010). 24. Palakodeti, A. et al. The molecular basis for modulation of human Vγ9Vδ2 T cell responses by CD277/butyrophilin‑3 (BTN3A)-specific antibodies. J. Biol. Chem. 287, 32780–32790 (2012). This paper provides an insight into the molecular basis of how different BTN3A‑specific monoclonal antibodies can drive disparate, and even opposing, biological functions.

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Acknowledgements

The authors would like to thank A. Gardet for critical reading of the manuscript.

Competing interests statement

The authors declare competing interests: see Web version for details.

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