Publisher Correction: γδ TCR ligands: the quest to solve a 500-million-year-old mystery.

In the version of this article initially published, the affiliations were incorrect. The correct affiliations are as follows: "1Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, UK. 2Institute of Immunology and Immunotherapy, Cancer Immunology and Immunotherapy Centre, Cancer Research UK Birmingham Centre, University of Birmingham, Birmingham, UK." The reference citation at the end of the first sentence of the second paragraph of the subsection 'A perspective on current methods of ligand identification' was incorrect; the correct citation is "...ligands20-40." There is an error (en dash) in the fourth paragraph of that section; the correct text is "...specific for CD1 and phycoerythrin...". There is an error ("proposed") in the fourth paragraph of the subsection 'An emerging adaptive perspective on antigenic γδ TCR ligands'; the correct text is "...are suggested to recognize...". There is an error ("via") in the fourth paragraph of the subsection 'B7-like molecules as candidate γδ TCR ligands'; the correct text is "...in a non-clonotypic fashion are striking...". Finally, reference citations throughout the legend to Fig. 1 are incorrect; the correct citations are as follows: MHC class I free heavy chain22; HLA-B580234; I-Ek (ref. 30); MSH2 (MutShomolog 2) and HSP60 (heat-shock protein 60)24; ULBP4 (UL16-binding protein 4)27; MICA41; the herpes simplex virus glycoprotein HSVgI33; ATPase-apolipoprotein-AI39; and insulin B:9-23 peptide antigen40. The errors have been corrected in the HTML and PDF versions of the article.

γδ TCR ligands: the quest to solve a 500-million-year-old mystery.

γδ T cells have been retained as a lineage over the majority of vertebrate evolution, are able to respond to immune challenges in unique ways, and are of increasing therapeutic interest. However, one central mystery has endured: the identity of the ligands recognized by the γδ T cell antigen receptor. Here we discuss the inherent challenges in answering this question, the new opportunities provided by recent studies, and the criteria by which the field might judge success.

Butyrophilin-2A1 Directly Binds Germline-Encoded Regions of the Vγ9Vδ2 TCR and Is Essential for Phosphoantigen Sensing.

Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.

Butyrophilin-like 3 Directly Binds a Human Vγ4+ T Cell Receptor Using a Modality Distinct from Clonally-Restricted Antigen.

Butyrophilin (BTN) and butyrophilin-like (BTNL/Btnl) heteromers are major regulators of human and mouse γδ T cell subsets, but considerable contention surrounds whether they represent direct γδ T cell receptor (TCR) ligands. We demonstrate that the BTNL3 IgV domain binds directly and specifically to a human Vγ4+ TCR, "LES" with an affinity (∼15-25 µM) comparable to many αß TCR-peptide major histocompatibility complex interactions. Mutations in germline-encoded Vγ4 CDR2 and HV4 loops, but not in somatically recombined CDR3 loops, drastically diminished binding and T cell responsiveness to BTNL3-BTNL8-expressing cells. Conversely, CDR3γ and CDR3δ loops mediated LES TCR binding to endothelial protein C receptor, a clonally restricted autoantigen, with minimal CDR1, CDR2, or HV4 contributions. Thus, the γδ TCR can employ two discrete binding modalities: a non-clonotypic, superantigen-like interaction mediating subset-specific regulation by BTNL/BTN molecules and CDR3-dependent, antibody-like interactions mediating adaptive γδ T cell biology. How these findings might broadly apply to γδ T cell regulation is also examined.

γδ TCR Recognition of MR1: Adapting to Life on the Flip Side.

Nonclassical class I MHC-like molecules are ligands for several unconventional T cell populations. Recently, Le Nours et al. identified human γδ T cells recognising MHC-related protein-1 (MR1) via their T cell receptor (TCR). Also recognised by the αß-TCR of mucosal associated invariant T cells, MR1 interacts with specific γδ-TCRs using strikingly diverse binding modes, suggesting fundamental differences in γδ T cell recognition.

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 V(γ)4V(δ)5 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 V(γ)4V(δ)5 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.

The distinct MHC-unrestricted immunobiology of innate-like and adaptive-like human γδ T cell subsets-Nature's CAR-T cells.

Distinct innate-like and adaptive-like immunobiological paradigms are emerging for human γδ T cells, supported by a combination of immunophenotypic, T cell receptor (TCR) repertoire, functional, and transcriptomic data. Evidence of the γδ TCR/ligand recognition modalities that respective human subsets utilize is accumulating. Although many questions remain unanswered, one superantigen-like modality features interactions of germline-encoded regions of particular TCR Vγ regions with specific BTN/BTNL family members and apparently aligns with an innate-like biology, albeit with some scope for clonal amplification. A second involves CDR3-mediated γδ TCR interaction with diverse ligands and aligns with an adaptive-like biology. Importantly, these unconventional modalities provide γδ T cells with unique recognition capabilities relative to αß T cells, B cells, and NK cells, allowing immunosurveillance for signatures of "altered self" on target cells, via a membrane-linked γδ TCR recognizing intact non-MHC proteins on the opposing cell surface. In doing so, they permit cellular responses in diverse situations including where MHC expression is compromised, or where conventional adaptive and/or NK cell-mediated immunity is suppressed. γδ T cells may therefore utilize their TCR like a cell-surface Fab repertoire, somewhat analogous to engineered chimeric antigen receptor T cells, but additionally integrating TCR signaling with parallel signals from other surface immunoreceptors, making them multimolecular sensors of cellular stress.

Development of a high-sensitivity ELISA detecting IgG, IgA and IgM antibodies to the SARS-CoV-2 spike glycoprotein in serum and saliva.

Detecting antibody responses during and after SARS-CoV-2 infection is essential in determining the seroepidemiology of the virus and the potential role of antibody in disease. Scalable, sensitive and specific serological assays are essential to this process. The detection of antibody in hospitalized patients with severe disease has proven relatively straightforward; detecting responses in subjects with mild disease and asymptomatic infections has proven less reliable. We hypothesized that the suboptimal sensitivity of antibody assays and the compartmentalization of the antibody response may contribute to this effect. We systematically developed an ELISA, optimizing different antigens and amplification steps, in serum and saliva from non-hospitalized SARS-CoV-2-infected subjects. Using trimeric spike glycoprotein, rather than nucleocapsid, enabled detection of responses in individuals with low antibody responses. IgG1 and IgG3 predominate to both antigens, but more anti-spike IgG1 than IgG3 was detectable. All antigens were effective for detecting responses in hospitalized patients. Anti-spike IgG, IgA and IgM antibody responses were readily detectable in saliva from a minority of RT-PCR confirmed, non-hospitalized symptomatic individuals, and these were mostly subjects who had the highest levels of anti-spike serum antibodies. Therefore, detecting antibody responses in both saliva and serum can contribute to determining virus exposure and understanding immune responses after SARS-CoV-2 infection.

Recasting Human Vδ1 Lymphocytes in an Adaptive Role.

γδ T cells are unconventional lymphocytes commonly described as 'innate-like' in function, which can respond in both a T cell receptor (TCR)-independent and also major histocompatibility complex (MHC)-unrestricted TCR-dependent manner. While the relative importance of TCR recognition had remained unclear, recent studies revealed that human Vδ1 T cells display unexpected parallels with adaptive αß T cells. Vδ1 T cells undergo profound and highly focussed clonal expansion from an initially diverse and private TCR repertoire, most likely in response to specific immune challenges. Concomitantly, they differentiate from a Vδ1 T cell naïve (Tnaïve) to a Vδ1 T cell effector (Teffector) phenotype, marked by the downregulation of lymphoid homing receptors and upregulation of peripheral homing receptors and effector markers. This suggests that an adaptive paradigm applies to Vδ1 T cells, likely involving TCR-dependent but MHC-unrestricted responses to microbial and non-microbial challenges.

Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for the presentation of transformed self.

Protein phosphorylation generates a source of phosphopeptides that are presented by major histocompatibility complex class I molecules and recognized by T cells. As deregulated phosphorylation is a hallmark of malignant transformation, the differential display of phosphopeptides on cancer cells provides an immunological signature of 'transformed self'. Here we demonstrate that phosphorylation can considerably increase peptide binding affinity for HLA-A2. To understand this, we solved crystal structures of four phosphopeptide-HLA-A2 complexes. These identified a novel peptide-binding motif centered on a solvent-exposed phosphate anchor. Our findings indicate that deregulated phosphorylation can create neoantigens by promoting binding to major histocompatibility complex molecules or by affecting the antigenic identity of presented epitopes. These results highlight the potential of phosphopeptides as novel targets for cancer immunotherapy.

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

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.

Human liver infiltrating γδ T cells are composed of clonally expanded circulating and tissue-resident populations.

BACKGROUND & AIMS: γδ T cells comprise a substantial proportion of tissue-associated lymphocytes. However, our current understanding of human γδ T cells is primarily based on peripheral blood subsets, while the immunobiology of tissue-associated subsets remains largely unclear. Therefore, we aimed to elucidate the T cell receptor (TCR) diversity, immunophenotype and function of γδ T cells in the human liver. METHODS: We characterised the TCR repertoire, immunophenotype and function of human liver infiltrating γδ T cells, by TCR sequencing analysis, flow cytometry, in situ hybridisation and immunohistochemistry. We focussed on the predominant tissue-associated Vδ2- γδ subset, which is implicated in liver immunopathology. RESULTS: Intrahepatic Vδ2- γδ T cells were highly clonally focussed, with single expanded clonotypes featuring complex, private TCR rearrangements frequently dominating the compartment. Such T cells were predominantly CD27lo/- effector lymphocytes, whereas naïve CD27hi, TCR-diverse populations present in matched blood were generally absent in the liver. Furthermore, while a CD45RAhi Vδ2- γδ effector subset present in both liver and peripheral blood contained overlapping TCR clonotypes, the liver Vδ2- γδ T cell pool also included a phenotypically distinct CD45RAlo effector compartment that was enriched for expression of the tissue tropism marker CD69, the hepatic homing chemokine receptors CXCR3 and CXCR6, and liver-restricted TCR clonotypes, suggestive of intrahepatic tissue residency. Liver infiltrating Vδ2- γδ cells were capable of polyfunctional cytokine secretion, and unlike peripheral blood subsets, were responsive to both TCR and innate stimuli. CONCLUSION: These findings suggest that the ability of Vδ2- γδ T cells to undergo clonotypic expansion and differentiation is crucial in permitting access to solid tissues, such as the liver, which results in functionally distinct peripheral and liver-resident memory γδ T cell subsets. They also highlight the inherent functional plasticity within the Vδ2- γδ T cell compartment and provide information that could be used for the design of cellular therapies that suppress liver inflammation or combat liver cancer. LAY SUMMARY: γδ T cells are frequently enriched in many solid tissues, however the immunobiology of such tissue-associated subsets in humans has remained unclear. We show that intrahepatic γδ T cells are enriched for clonally expanded effector T cells, whereas naïve γδ T cells are largely excluded. Moreover, whereas a distinct proportion of circulating T cell clonotypes was present in both the liver tissue and peripheral blood, a functionally and clonotypically distinct population of liver-resident γδ T cells was also evident. Our findings suggest that factors triggering γδ T cell clonal selection and differentiation, such as infection, can drive enrichment of γδ T cells into liver tissue, allowing the development of functionally distinct tissue-restricted memory populations specialised in local hepatic immunosurveillance.

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.

Structural and energetic evidence for highly peptide-specific tumor antigen targeting via allo-MHC restriction.

Immunotherapies targeting peptides presented by allogeneic MHC molecules offer the prospect of circumventing tolerance to key tumor-associated self-antigens. However, the degree of antigen specificity mediated by alloreactive T cells, and their ability to discriminate normal tissues from transformed cells presenting elevated antigen levels, is poorly understood. We examined allorecognition of an HLA-A2-restricted Hodgkin's lymphoma-associated antigen and were able to isolate functionally antigen-specific allo-HLA-A2-restricted T cells from multiple donors. Binding and structural studies, focused on a prototypic allo-HLA-A2-restricted T-cell receptor (TCR) termed NB20 derived from an HLA-A3 homozygote, suggested highly peptide-specific allorecognition that was energetically focused on antigen, involving direct recognition of a distinct allopeptide presented within a conserved MHC recognition surface. Although NB20/HLA-A2 affinity was unremarkable, TCR/MHC complexes were very short-lived, consistent with suboptimal TCR triggering and tolerance to low antigen levels. These data provide strong molecular evidence that within the functionally heterogeneous alloreactive repertoire, there is the potential for highly antigen-specific "allo-MHC-restricted" recognition and suggest a kinetic mechanism whereby allo-MHC-restricted T cells may discriminate normal from transformed tissue, thereby outlining a suitable basis for broad-based therapeutic targeting of tolerizing tumor antigens.

Phosphonodiamidate prodrugs of phosphoantigens (ProPAgens) exhibit potent Vγ9/Vδ2 T cell activation and eradication of cancer cells.

The phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) is an established activator of Vγ9/Vδ2 T cells and stimulates downstream effector functions including cytotoxicity and cytokine production. In order to improve its drug-like properties, we herein report the design, synthesis, serum stability, in vitro metabolism, and biological evaluation of a new class of symmetrical phosphonodiamidate prodrugs of methylene and difluoromethylene monophosphonate derivatives of HMBPP. These prodrugs, termed phosphonodiamidate ProPAgens, were synthesized in good yields, exhibited excellent serum stability (>7 h), and their in vitro metabolism was shown to be initiated by carboxypeptidase Y. These phosphonodiamidate ProPAgens triggered potent activation of Vγ9/Vδ2 T cells, which translated into efficient Vγ9/Vδ2 T cell-mediated eradication of bladder cancer cells in vitro. Together, these findings showcase the potential of these phosphonodiamidate ProPAgens as Vγ9/Vδ2 T cell modulators that could be further developed as novel cancer immunotherapeutic agents.

Modifications outside CDR1, 2 and 3 of the TCR variable ß domain increase TCR expression and antigen-specific function.

T cell receptor (TCR) gene modified T cells are a promising form of adoptive cellular therapy against human malignancies and viral infections. Since the first human clinical trial was carried out in 2006, several strategies have been developed to improve the efficacy and safety of TCR engineered T cells by enhancing the surface expression of the introduced therapeutic TCRs whilst reducing the mis-pairing with endogenous TCR chains. In this study, we explored how modifications of framework residues in the TCR variable domains affect TCR expression and function. We used bioinformatic and protein structural analyses to identify candidate amino acid residues in the framework of the variable ß domain predicted to drive high TCR surface expression. Changes of these residues in poorly expressed TCRs resulted in improved surface expression and boosted target cell specific killing by engineered T cells expressing the modified TCRs. Overall, these results indicate that small changes in the framework of the TCR variable domains can result in improved expression and functionality, while at the same time reducing the risk of toxicity associated with TCR mis-pairing.