Carrie R. Willcox

Cytomegalovirus and tumor stress surveillance by binding of a human γδ T cell antigen receptor to endothelial protein C receptor.

T cells bearing γδ T cell antigen receptors (TCRs) function in lymphoid stress surveillance. However, the contribution of γδ TCRs to such responses is unclear. Here we found that the TCR of a human Vγ4Vδ5 clone directly bound endothelial protein C receptor (EPCR), which allowed γδ T cells to recognize both endothelial cells targeted by cytomegalovirus and epithelial tumors. EPCR is a major histocompatibility complex–like molecule that binds lipids analogously to the antigen-presenting molecule CD1d. However, the Vγ4Vδ5 TCR bound EPCR independently of lipids, in an antibody-like way. Moreover, the recognition of target cells by γδ T cells required a multimolecular stress signature composed of EPCR and costimulatory ligand(s). Our results demonstrate how a γδ TCR mediates recognition of broadly stressed human cells by engaging a stress-regulated self antigen.

A mycolic acid–specific CD1-restricted T cell population contributes to acute and memory immune responses in human tuberculosis infection

Current tuberculosis (TB) vaccine strategies are largely aimed at activating conventional T cell responses to mycobacterial protein antigens. However, the lipid-rich cell wall of Mycobacterium tuberculosis (M. tuberculosis) is essential for pathogenicity and provides targets for unconventional T cell recognition. Group 1 CD1-restricted T cells recognize mycobacterial lipids, but their function in human TB is unclear and their ability to establish memory is unknown. Here, we characterized T cells specific for mycolic acid (MA), the predominant mycobacterial cell wall lipid and key virulence factor, in patients with active TB infection. MA-specific T cells were predominant in TB patients at diagnosis, but were absent in uninfected bacillus Calmette-Guérin-vaccinated (BCG-vaccinated) controls. These T cells were CD1b restricted, detectable in blood and disease sites, produced both IFN-γ and IL-2, and exhibited effector and central memory phenotypes. MA-specific responses contracted markedly with declining pathogen burden and, in patients followed longitudinally, exhibited recall expansion upon antigen reencounter in vitro long after successful treatment, indicative of lipid-specific immunological memory. T cell recognition of MA is therefore a significant component of the acute adaptive and memory immune response in TB, suggesting that mycobacterial lipids may be promising targets for improved TB vaccines.

Immunological Visibility: Posttranscriptional Regulation of Human NKG2D Ligands by the EGF Receptor Pathway

Environmental changes induce human NKG2D ligand expression—immunological visibility—through the EGFR stress response. Attracting Big Brother It is increasingly acknowledged that immune cells serve as the surveillance system of the body—constantly on the lookout for things that are out of place, such as infections or cancer. Indeed, researchers and clinicians are now trying to harness the immune system either by overcoming inhibitions or by pointing them at particular targets. But what happens when the target tries to hide? Vantourout et al. now look at the mechanisms that regulate immunological visibility. The authors stressed human epithelial cells by various means and found that up-regulation of ligands that activate NKG2D, a lymphocyte-activating receptor, was dependent on epithelial growth factor receptor (EGFR). They looked further into the mechanism and discovered that EGFR activation resulted in relocalization of AUF1 proteins, which normally destabilize NKG2D ligands, and resulted in increased expression of these ligands—and improved immunological visibility. What’s more, EGFR activation is one of the most common forms of dysregulation underpinning human carcinomas, and primary tumors with high EGFR expression showed higher NKG2D ligand expression levels. Conversely, NKG2D ligand expression was reduced by erlotinib and cetuximab, two EGFR inhibitors commonly used in the clinic. These data suggest that the effects of therapeutics that limit cancer growth should be considered with immunotherapy. Human cytolytic T lymphocytes and natural killer cells can limit tumor growth and are being increasingly harnessed for tumor immunotherapy. One way cytolytic lymphocytes recognize tumor cells is by engagement of their activating receptor, NKG2D, by stress antigens of the MICA/B and ULBP families. This study shows that surface up-regulation of NKG2D ligands by human epithelial cells in response to ultraviolet irradiation, osmotic shock, oxidative stress, and growth factor provision is attributable to activation of the epidermal growth factor receptor (EGFR). EGFR activation causes intracellular relocalization of AUF1 proteins that ordinarily destabilize NKG2D ligand mRNAs by targeting an AU-rich element conserved within the 3′ ends of most human, but not murine, NKG2D ligand genes. Consistent with these findings, NKG2D ligand expression by primary human carcinomas positively correlated with EGFR expression, which is commonly hyperactivated in such tumors, and was reduced by clinical EGFR inhibitors. Therefore, stress-induced activation of EGFR not only regulates cell growth but also concomitantly regulates the cells’ immunological visibility. Thus, therapeutics designed to limit cancer cell growth should also be considered in terms of their impact on immunosurveillance.

Clonal selection in the human Vδ1 T cell repertoire indicates γδ TCR-dependent adaptive immune surveillance

γδ T cells are considered to be innate-like lymphocytes that respond rapidly to stress without clonal selection and differentiation. Here we use next-generation sequencing to probe how this paradigm relates to human Vδ2neg T cells, implicated in responses to viral infection and cancer. The prevalent Vδ1 T cell receptor (TCR) repertoire is private and initially unfocused in cord blood, typically becoming strongly focused on a few high-frequency clonotypes by adulthood. Clonal expansions have differentiated from a naive to effector phenotype associated with CD27 downregulation, retaining proliferative capacity and TCR sensitivity, displaying increased cytotoxic markers and altered homing capabilities, and remaining relatively stable over time. Contrastingly, Vδ2+ T cells express semi-invariant TCRs, which are present at birth and shared between individuals. Human Vδ1+ T cells have therefore evolved a distinct biology from the Vδ2+ subset, involving a central, personalized role for the γδ TCR in directing a highly adaptive yet unconventional form of immune surveillance.

Sensing of cell stress by human γδ TCR-dependent recognition of annexin A2

Significance Human γδ T lymphocytes have innate-like and adaptive-like functions and can circulate in blood or reside in tissues. They are activated by specific antigens recognized by their T-cell receptor and recognize infected and transformed cells, suggesting that cellular stress is involved in specific antigen expression. However, molecular characterization of stress-induced antigens remains elusive, hampering our understanding of the role of γδ T cells in cancer and infections. In the present study we identify annexin A2 as such stress-induced antigen known as a phospholipid-binding protein involved in tumorigenesis, redox potential regulation, and wound healing. Stress-mediated membrane exposure of annexin A2 could thus constitute a danger signal for γδ T cells to recognize various cell dysregulations and protect the host against cancer and infections. Human γδ T cells comprise a first line of defense through T-cell receptor (TCR) recognition of stressed cells. However, the molecular determinants and stress pathways involved in this recognition are largely unknown. Here we show that exposure of tumor cells to various stress situations led to tumor cell recognition by a Vγ8Vδ3 TCR. Using a strategy that we previously developed to identify antigenic ligands of γδ TCRs, annexin A2 was identified as the direct ligand of Vγ8Vδ3 TCR, and was found to be expressed on tumor cells upon the stress situations tested in a reactive oxygen species-dependent manner. Moreover, purified annexin A2 was able to stimulate the proliferation of a Vδ2neg γδ T-cell subset within peripheral blood mononuclear cells and other annexin A2-specific Vδ2neg γδ T-cell clones could be derived from peripheral blood mononuclear cells. We thus propose membrane exposure of annexin A2 as an oxidative stress signal for some Vδ2neg γδ T cells that could be involved in an adaptive stress surveillance.

BTN3A1 discriminates γδ T cell phosphoantigens from non-antigenic small molecules via a conformational sensor in its B30.2 domain

Human Vγ9/Vδ2 T-cells detect tumor cells and microbial infections by recognizing small phosphorylated prenyl metabolites termed phosphoantigens (P-Ag). The type-1 transmembrane protein Butyrophilin 3A1 (BTN3A1) is critical to the P-Ag-mediated activation of Vγ9/Vδ2 T-cells; however, the molecular mechanisms involved in BTN3A1-mediated metabolite sensing are unclear, including how P-Ags are discriminated from nonantigenic small molecules. Here, we utilized NMR and X-ray crystallography to probe P-Ag sensing by BTN3A1. Whereas the BTN3A1 immunoglobulin variable domain failed to bind P-Ag, the intracellular B30.2 domain bound a range of negatively charged small molecules, including P-Ag, in a positively charged surface pocket. However, NMR chemical shift perturbations indicated BTN3A1 discriminated P-Ag from nonantigenic small molecules by their ability to induce a specific conformational change in the B30.2 domain that propagated from the P-Ag binding site to distal parts of the domain. These results suggest BTN3A1 selectively detects P-Ag intracellularly via a conformational antigenic sensor in its B30.2 domain and have implications for rational design of antigens for Vγ9/Vδ2-based T-cell immunotherapies.

A disease-linked ULBP6 polymorphism inhibits NKG2D-mediated target cell killing by enhancing the stability of NKG2D ligand binding

A disease-associated variant of an activating ligand engages receptors so strongly that it impairs NK cell–mediated killing. Distracting natural killer cells Natural killer (NK) cells target virally infected and transformed cells for cytolysis. When sufficient activating receptors on the NK cell surface, such as NKG2D, are engaged by ligands on the target cell, such as ULBP proteins, the NK cell kills the target. Polymorphisms within ULBP-encoding genes are associated with immune dysfunction. Zuo et al. found that the affinity of a commonly occurring ULBP6 variant for NKG2D was greater than that of the wild-type protein, which impaired NK cell activation. A soluble form of this protein variant bound so tightly to NKG2D that it suppressed receptor activation and target cell killing in response to other NKG2D ligands. Together, these data suggest that targeting NK cell–ligand interactions may provide therapies to modulate the strength of immune responses. NKG2D (natural killer group 2, member D) is an activating receptor found on the surface of immune cells, including natural killer (NK) cells, which regulates innate and adaptive immunity through recognition of the stress-induced ligands ULBP1 (UL16 binding protein 1) to ULBP6 and MICA/B. Similar to class I human leukocyte antigen (HLA), these NKG2D ligands have a major histocompatibility complex–like fold and exhibit pronounced polymorphism, which influences human disease susceptibility. However, whereas class I HLA polymorphisms occur predominantly in the α1α2 groove and affect antigen binding, the effects of most NKG2D ligand polymorphisms are unclear. We studied the molecular and functional consequences of the two major alleles of ULBP6, the most polymorphic ULBP gene, which are associated with autoimmunity and relapse after stem cell transplantation. Surface plasmon resonance and crystallography studies revealed that the arginine-to-leucine polymorphism within ULBP0602 affected the NKG2D-ULBP6 interaction by generating an energetic hotspot. This resulted in an NKG2D-ULBP0602 affinity of 15.5 nM, which is 10- to 1000-fold greater than the affinities of other ULBP-NKG2D interactions and limited NKG2D-mediated activation. In addition, soluble ULBP0602 exhibited high-affinity competitive binding for NKG2D and partially suppressed NKG2D-mediated activation of NK cells by other NKG2D ligands. These effects resulted in a decrease in a range of NKG2D-mediated effector functions. Our results reveal that ULBP polymorphisms affect the strength of human lymphocyte responses to cellular stress signals and may offer opportunities for therapeutic intervention.

Resolving the mystery of pyrophosphate antigen presentation

Small phosphorylated prenyl metabolites are potent activators of γδ T cells in human peripheral blood, but the molecular mechanism underlying their antigenic potency has remained a mystery. New data identify BTN3A1 as a novel antigen-presentation molecule for both microbial and host-derived phosphorylated antigens.

The human Vδ2 + T-cell compartment comprises distinct innate-like Vγ9 + and adaptive Vγ9 - subsets

Vδ2+ T cells form the predominant human γδ T-cell population in peripheral blood and mediate T-cell receptor (TCR)-dependent anti-microbial and anti-tumour immunity. Here we show that the Vδ2+ compartment comprises both innate-like and adaptive subsets. Vγ9+ Vδ2+ T cells display semi-invariant TCR repertoires, featuring public Vγ9 TCR sequences equivalent in cord and adult blood. By contrast, we also identify a separate, Vγ9− Vδ2+ T-cell subset that typically has a CD27hiCCR7+CD28+IL-7Rα+ naive-like phenotype and a diverse TCR repertoire, however in response to viral infection, undergoes clonal expansion and differentiation to a CD27loCD45RA+CX3CR1+granzymeA/B+ effector phenotype. Consistent with a function in solid tissue immunosurveillance, we detect human intrahepatic Vγ9− Vδ2+ T cells featuring dominant clonal expansions and an effector phenotype. These findings redefine human γδ T-cell subsets by delineating the Vδ2+ T-cell compartment into innate-like (Vγ9+) and adaptive (Vγ9−) subsets, which have distinct functions in microbial immunosurveillance.Human Vδ2+ γδ T cells are thought to be an innate-like T-cell population. Here the authors show the Vδ2+ compartment contains both innate-like Vγ9+ and an adaptive Vγ9- subset that undergoes clonal expansion during viral infection and can infiltrate liver tissue.

Characterization of a Putative Receptor Binding Surface on Skint-1, a Critical Determinant of Dendritic Epidermal T Cell Selection

Dendritic epidermal T cells (DETC) form a skin-resident γδ T cell population that makes key contributions to cutaneous immune stress surveillance, including non-redundant contributions to protection from cutaneous carcinogens. How DETC become uniquely associated with the epidermis was in large part solved by the identification of Skint-1, the prototypic member of a novel B7-related multigene family. Expressed only by thymic epithelial cells and epidermal keratinocytes, Skint-1 drives specifically the development of DETC progenitors, making it the first clear candidate for a selecting ligand for non-MHC/CD1-restricted T cells. However, the molecular mechanisms underpinning Skint-1 activity are unresolved. Here, we provide evidence that DETC selection requires Skint-1 expression on the surface of thymic epithelial cells, and depends upon specific residues on the CDR3-like loop within the membrane-distal variable domain of Skint-1 (Skint-1 DV). Nuclear magnetic resonance of Skint-1 DV revealed a core tertiary structure conserved across the Skint family, but a highly distinct surface charge distribution, possibly explaining its unique function. Crucially, the CDR3-like loop formed an electrostatically distinct surface, featuring key charged and hydrophobic solvent-exposed residues, at the membrane-distal tip of DV. These results provide the first structural insights into the Skint family, identifying a putative receptor binding surface that directly implicates Skint-1 in receptor-ligand interactions crucial for DETC selection.