Btla signaling in conventional and regulatory lymphocytes coordinately tempers humoral immunity in the intestinal mucosa.

The Btla inhibitory receptor limits innate and adaptive immune responses, both preventing the development of autoimmune disease and restraining anti-viral and anti-tumor responses. It remains unclear how the functions of Btla in diverse lymphocytes contribute to immunoregulation. Here, we show that Btla inhibits activation of genes regulating metabolism and cytokine signaling, including Il6 and Hif1a, indicating a regulatory role in humoral immunity. Within mucosal Peyer's patches, we find T-cell-expressed Btla-regulated Tfh cells, while Btla in T or B cells regulates GC B cell numbers. Treg-expressed Btla is required for cell-intrinsic Treg homeostasis that subsequently controls GC B cells. Loss of Btla in lymphocytes results in increased IgA bound to intestinal bacteria, correlating with altered microbial homeostasis and elevations in commensal and pathogenic bacteria. Together our studies provide important insights into how Btla functions as a checkpoint in diverse conventional and regulatory lymphocyte subsets to influence systemic immune responses.

HVEM network signaling in cancer.

Somatic mutations in cancer cells may influence tumor growth, survival, or immune interactions in their microenvironment. The tumor necrosis factor receptor family member HVEM (TNFRSF14) is frequently mutated in cancers and has been attributed a tumor suppressive role in some cancer contexts. HVEM functions both as a ligand for the lymphocyte checkpoint proteins BTLA and CD160, and as a receptor that activates NF-κB signaling pathways in response to BTLA and CD160 and the TNF ligands LIGHT and LTα. BTLA functions to inhibit lymphocyte activation, but has also been ascribed a role in stimulating cell survival. CD160 functions to co-stimulate lymphocyte function, but has also been shown to activate inhibitory signaling in CD4+ T cells. Thus, the role of HVEM within diverse cancers and in regulating the immune responses to these tumors is likely context specific. Additionally, development of therapeutics that target proteins within this network of interacting proteins will require a deeper understanding of how these proteins function in a cancer-specific manner. However, the prominent role of the HVEM network in anti-cancer immune responses indicates a promising area for drug development.

A herpesvirus entry mediator mutein with selective agonist action for the inhibitory receptor B and T lymphocyte attenuator.

The human cytomegalovirus opening reading frame UL144 is an ortholog of the TNF receptor superfamily member, herpesvirus entry mediator (HVEM; TNFRSF14). HVEM binds the TNF ligands, LIGHT and LTa; the immunoglobulin inhibitory receptor, B and T lymphocyte attenuator (BTLA); and the natural killer cell-activating receptor CD160. However, UL144 selectively binds BTLA, avoiding activation of inflammatory signaling initiated by CD160 in natural killer cells. BTLA and CD160 cross-compete for binding HVEM, but the structural basis for the ligand selectivity by UL144 and how it acts as an anti-inflammatory agonist remains unclear. Here, we modeled the UL144 structure and characterized its binding with BTLA. The UL144 structure was predicted to closely mimic the surface of HVEM, and we also found that both HVEM and UL144 bind a common epitope of BTLA, whether engaged in trans or in cis, that is shared with a BTLA antibody agonist. On the basis of the UL144 selectivity, we engineered a BTLA-selective HVEM protein to understand the basis for ligand selectivity and BTLA agonism to develop novel anti-inflammatory agonists. This HVEM mutein did not bind CD160 or TNF ligands but did bind BTLA with 10-fold stronger affinity than wild-type HVEM and retained potent inhibitory activity that reduced T-cell receptor, B-cell receptor, and interferon signaling in B cells. In conclusion, using a viral immune evasion strategy that shows broad immune-ablating activity, we have identified a novel anti-inflammatory BTLA-selective agonist.

The TNF Receptor Superfamily in Co-stimulating and Co-inhibitory Responses.

Cytokines related to tumor necrosis factor (TNF) provide a communication network essential for coordinating multiple cell types into an effective host defense system against pathogens and malignant cells. The pathways controlled by the TNF superfamily differentiate both innate and adaptive immune cells and modulate stromal cells into microenvironments conducive to host defenses. Members of the TNF receptor superfamily activate diverse cellular functions from the production of type 1 interferons to the modulation of survival of antigen-activated T cells. Here, we focus attention on the subset of TNF superfamily receptors encoded in the immune response locus in chromosomal region 1p36. Recent studies have revealed that these receptors use diverse mechanisms to either co-stimulate or restrict immune responses. Translation of the fundamental mechanisms of TNF superfamily is leading to the design of therapeutics that can alter pathogenic processes in several autoimmune diseases or promote immunity to tumors.

Human CD4+CD3- innate-like T cells provide a source of TNF and lymphotoxin-αß and are elevated in rheumatoid arthritis.

Innate lymphoid cells encompass a diverse array of lymphocyte subsets with unique phenotype that initiate inflammation and provide host defenses in specific microenvironments. In this study, we identify a rare human CD4(+)CD3(-) innate-like lymphoid population with high TNF expression that is enriched in blood from patients with rheumatoid arthritis. These CD4(+)CD3(-) cells belong to the T cell lineage, but the lack of AgR at the cell surface renders them nonresponsive to TCR-directed stimuli. By developing a culture system that sustains survival, we show that CD4(+)CD3(-) innate-like T cells display IL-7-dependent induction of surface lymphotoxin-αß, demonstrating their potential to modify tissue microenvironments. Furthermore, expression of CCR6 on the CD4(+)CD3(-) population defines a CD127(high) subset that is highly responsive to IL-7. This CD4(+)CD3(-) population is enriched in the peripheral blood from rheumatoid arthritis patients, suggesting a link to their involvement in chronic inflammatory disease.

CD160 activation by herpesvirus entry mediator augments inflammatory cytokine production and cytolytic function by NK cells.

Lymphocyte activation is regulated by costimulatory and inhibitory receptors, of which both B and T lymphocyte attenuator (BTLA) and CD160 engage herpesvirus entry mediator (HVEM). Notably, it remains unclear how HVEM functions with each of its ligands during immune responses. In this study, we show that HVEM specifically activates CD160 on effector NK cells challenged with virus-infected cells. Human CD56(dim) NK cells were costimulated specifically by HVEM but not by other receptors that share the HVEM ligands LIGHT, Lymphotoxin-α, or BTLA. HVEM enhanced human NK cell activation by type I IFN and IL-2, resulting in increased IFN-γ and TNF-α secretion, and tumor cell-expressed HVEM activated CD160 in a human NK cell line, causing rapid hyperphosphorylation of serine kinases ERK1/2 and AKT and enhanced cytolysis of target cells. In contrast, HVEM activation of BTLA reduced cytolysis of target cells. Together, our results demonstrate that HVEM functions as a regulator of immune function that activates NK cells via CD160 and limits lymphocyte-induced inflammation via association with BTLA.

TNF Superfamily Networks: bidirectional and interference pathways of the herpesvirus entry mediator (TNFSF14).

The herpesvirus entry mediator (HVEM; TNFRSF14) can activate either proinflammatory or inhibitory signaling pathways. HVEM engages two distinct types of ligands, the canonical TNF-related cytokines, LIGHT and Lymphotoxin-α, and the Ig-related membrane proteins, BTLA (B and T lymphocyte attenuator) and CD160. Recent evidence indicates that the signal generated by HVEM depends on the context of its ligands expressed in trans or in cis. HVEM engagement by all of its ligands in trans initiates bidirectional signaling. In contrast, naïve T cells coexpress BTLA and HVEM forming a cis-complex that interferes with the activation of HVEM by extraneous ligands in the surrounding microenvironment. The HVEM Network is emerging as a key survival system for effector and memory T cells in mucosal tissues.

Cross-regulation between herpesviruses and the TNF superfamily members.

Herpesviruses have evolved numerous strategies to subvert host immune responses so they can coexist with their host species. These viruses 'co-opt' host genes for entry into host cells and then express immunomodulatory genes, including mimics of members of the tumour-necrosis factor (TNF) superfamily, that initiate and alter host-cell signalling pathways. TNF superfamily members have crucial roles in controlling herpesvirus infection by mediating the direct killing of infected cells and by enhancing immune responses. Despite these strong immune responses, herpesviruses persist in a latent form, which suggests a dynamic relationship between the host immune system and the virus that results in a balance between host survival and viral control.

Structural determinants of herpesvirus entry mediator recognition by murine B and T lymphocyte attenuator.

The B and T lymphocyte attenuator (BTLA) appears to act as a negative regulator of T cell activation and growth. BTLA specifically interacts with herpesvirus entry mediator (HVEM), a member of the TNFR family. Herein, we have undertaken surface plasmon resonance studies to quantitatively assess BTLA and HVEM ectodomain interactions. We find that soluble BALB/cJ BTLA engages HVEM with an equilibrium affinity of 0.97+/-0.19 microM while the C57BL/6 BTLA binds slightly better with an equilibrium affinity of 0.42+/-0.06 microM. Despite its lower affinity for HVEM, the kinetic half-life of BALB/cJ BTLA complexes are twice as long as observed for C57BL/6 BTLA (4 vs 2 s). To further explore these interactions, we solved the crystal structure of a murine BTLA (BALB/cJ) ectodomain at 1.8-A resolution, revealing a beta sandwich fold with strong similarity to I-set members of the Ig superfamily. Using a structure-based mutagenesis strategy, we then examined the individual contributions of 26 BTLA surface-exposed residues toward HVEM binding. Four single-site substitutions were identified that decrease HVEM binding below detectable levels and two that decrease binding by more than half. All six of these cluster at the edge of the beta sandwich in a membrane distal patch formed primarily from the A and G strands. This patch falls within the contacting surface recently revealed in the crystal structure of the human BTLA-HVEM cocomplex. The critical binding residues identified here are highly conserved across species, suggesting that BTLA employs a conserved binding mode for HVEM recognition.

Unexpected role of B and T lymphocyte attenuator in sustaining cell survival during chronic allostimulation.

B and T lymphocyte attenuator (BTLA; CD272) can deliver inhibitory signals to B and T cells upon binding its ligand herpesvirus entry mediator. Because CD28, CTLA-4, programmed death-1, and ICOS regulate the development of acute graft-vs-host disease (GVHD), we wished to assess if BTLA also played a role in this T cell-mediated response. In the nonirradiated parental-into-F1 model of acute GVHD, BTLA+/+ and BTLA-/- donor lymphocytes showed equivalent engraftment and expansion during the first week of the alloresponse. Unexpectedly, BTLA-/- donor T cells failed to sustain GVHD, showing a decline in surviving donor cell numbers beginning at day 9 and greatly reduced by day 11. Similarly, inhibition of BTLA-herpesvirus entry mediator engagement by in vivo administration of a blocking anti-BTLA Ab also caused reduced survival of donor cells. Microarray analysis revealed several genes that were differentially expressed by BTLA-/- and BTLA+/+ donor CD4+ T cells preceding the decline in BTLA-/- donor T cells. Several genes influencing Th cell polarization were differentially expressed by BTLA+/+ and BTLA-/- donor cells. Additionally, the re-expression of the IL-7Ralpha subunit that occurs in BTLA+/+ donor cells after 1 wk of in vivo allostimulation was not observed in BTLA-/- donor CD4+ cells. The striking loss of BTLA-/- T cells in this model indicates a role for BTLA activity in sustaining CD4+ T cell survival under the conditions of chronic stimulation in the nonirradiated parental-into-F1 GVHD.

Balancing co-stimulation and inhibition with BTLA and HVEM.

The interaction between B- and T-lymphocyte attenuator (BTLA), an inhibitory receptor whose extracellular domain belongs to the immunoglobulin superfamily, and herpesvirus-entry mediator (HVEM), a co-stimulatory tumour-necrosis factor receptor, is unique in that it is the only receptor-ligand interaction that directly bridges these two families of receptors. This interaction has raised many questions about how receptors from two different families could interact and what downstream signalling events might occur as a result of receptor ligation. As we discuss, recent studies show that engagement of HVEM with its endogenous ligand (LIGHT) from the tumour-necrosis factor family induces a powerful immune response, whereas HVEM interactions with BTLA negatively regulate T-cell responses.

B and T lymphocyte attenuator exhibits structural and expression polymorphisms and is highly Induced in anergic CD4+ T cells.

B and T lymphocyte attenuator (BTLA) was initially identified as expressed on Th1 cells and B cells, but recently reported to be expressed by macrophages, dendritic cells, and NK cells as well. To address this discrepancy we generated a panel of BTLA-specific mAbs and characterized BTLA expression under various activation conditions. We report the existence of three distinct BTLA alleles among 23 murine strains, differing both in Ig domain structure and cellular distribution of expression on lymphoid subsets. The BALB/c and MRL/lpr alleles differ at one amino acid residue, but C57BL/6 has nine additional differences and alters the predicted cysteine bonding pattern. The BALB/c BTLA allele is also expressed by B cells, T cells, and dendritic cells, but not macrophages or NK cells. However, C57BL/6 BTLA is expressed on CD11b+ macrophages and NK cells. Finally, in CD4+ T cells, BTLA is expressed most highly following Ag-specific induction of anergy in vivo, and unlike programmed death-1 and CTLA-4, not expressed by CD25+ regulatory T cells. These results clarify discrepancies regarding BTLA expression, suggest that structural and expression polymorphisms be considered when analyzing BTLA in various murine backgrounds, and indicate a possible role in anergic CD4+ T cells.

B and T lymphocyte attenuator regulates T cell activation through interaction with herpesvirus entry mediator.

B and T lymphocyte attenuator (BTLA) provides an inhibitory signal to B and T cells. Previously, indirect observations suggested that B7x was a ligand for BTLA. Here we show that BTLA does not bind B7x; instead, we identify herpesvirus entry mediator (HVEM) as the unique BTLA ligand. BTLA bound the most membrane-distal cysteine-rich domain of HVEM, distinct from regions where the ligands LIGHT and lymphotoxin-alpha bound HVEM. HVEM induced BTLA tyrosine phosphorylation and association of the tyrosine phosphatase SHP-2 and repressed antigen-driven T cell proliferation, providing an example of reverse signaling to a non-tumor necrosis factor family ligand. The conservation of the BTLA-HVEM interaction between mouse and human suggests that this system is an important pathway regulating lymphocyte activation and/or homeostasis in the immune response.

BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1.

During activation, T cells express receptors for receiving positive and negative costimulatory signals. Here we identify the B and T lymphocyte attenuator (BTLA), an immunoglobulin domain-containing glycoprotein with two immunoreceptor tyrosine-based inhibitory motifs. BTLA is not expressed by naive T cells, but it is induced during activation and remains expressed on T helper type 1 (T(H)1) but not T(H)2 cells. Crosslinking BTLA with antigen receptors induces its tyrosine phosphorylation and association with the Src homology domain 2 (SH2)-containing protein tyrosine phosphatases SHP-1 and SHP-2, and attenuates production of interleukin 2 (IL-2). BTLA-deficient T cells show increased proliferation, and BTLA-deficient mice have increased specific antibody responses and enhanced sensitivity to experimental autoimmune encephalomyelitis. B7x, a peripheral homolog of B7, is a ligand of BTLA. Thus, BTLA is a third inhibitory receptor on T lymphocytes with similarities to cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1).