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.

A distinct topology of BTN3A IgV and B30.2 domains controlled by juxtamembrane regions favors optimal human γδ T cell phosphoantigen sensing.

Butyrophilin (BTN)-3A and BTN2A1 molecules control the activation of human Vγ9Vδ2 T cells during T cell receptor (TCR)-mediated sensing of phosphoantigens (PAg) derived from microbes and tumors. However, the molecular rules governing PAg sensing remain largely unknown. Here, we establish three mechanistic principles of PAg-mediated γδ T cell activation. First, in humans, following PAg binding to the intracellular BTN3A1-B30.2 domain, Vγ9Vδ2 TCR triggering involves the extracellular V-domain of BTN3A2/BTN3A3. Moreover, the localization of both protein domains on different chains of the BTN3A homo-or heteromers is essential for efficient PAg-mediated activation. Second, the formation of BTN3A homo-or heteromers, which differ in intracellular trafficking and conformation, is controlled by molecular interactions between the juxtamembrane regions of the BTN3A chains. Finally, the ability of PAg not simply to bind BTN3A-B30.2, but to promote its subsequent interaction with the BTN2A1-B30.2 domain, is essential for T-cell activation. Defining these determinants of cooperation and the division of labor in BTN proteins improves our understanding of PAg sensing and elucidates a mode of action that may apply to other BTN family members.

Activated tissue resident memory T-cells (CD8+CD103+CD39+) uniquely predict survival in left sided "immune-hot" colorectal cancers.

Introduction: Characterization of the tumour immune infiltrate (notably CD8+ T-cells) has strong predictive survival value for cancer patients. Quantification of CD8 T-cells alone cannot determine antigenic experience, as not all infiltrating T-cells recognize tumour antigens. Activated tumour-specific tissue resident memory CD8 T-cells (TRM) can be defined by the co-express of CD103, CD39 and CD8. We investigated the hypothesis that the abundance and localization of TRM provides a higher-resolution route to patient stratification. Methods: A comprehensive series of 1000 colorectal cancer (CRC) were arrayed on a tissue microarray, with representative cores from three tumour locations and the adjacent normal mucosa. Using multiplex immunohistochemistry we quantified and determined the localization of TRM. Results: Across all patients, activated TRM were an independent predictor of survival, and superior to CD8 alone. Patients with the best survival had immune-hot tumours heavily infiltrated throughout with activated TRM. Interestingly, differences between right- and left-sided tumours were apparent. In left-sided CRC, only the presence of activated TRM (and not CD8 alone) was prognostically significant. Patients with low numbers of activated TRM cells had a poor prognosis even with high CD8 T-cell infiltration. In contrast, in right-sided CRC, high CD8 T-cell infiltration with low numbers of activated TRM was a good prognosis. Conclusion: The presence of high intra-tumoural CD8 T-cells alone is not a predictor of survival in left-sided CRC and potentially risks under treatment of patients. Measuring both high tumour-associated TRM and total CD8 T-cells in left-sided disease has the potential to minimize current under-treatment of patients. The challenge will be to design immunotherapies, for left-sided CRC patients with high CD8 T-cells and low activate TRM,that result in effective immune responses and thereby improve patient survival.

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.

Division of labor and cooperation between different butyrophilin proteins controls phosphoantigen-mediated activation of human γδ T cells.

Butyrophilin (BTN)-3A and BTN2A1 molecules control TCR-mediated activation of human Vγ9Vδ2 T-cells triggered by phosphoantigens (PAg) from microbes and tumors, but the molecular rules governing antigen sensing are unknown. Here we establish three mechanistic principles of PAg-action. Firstly, in humans, following PAg binding to the BTN3A1-B30.2 domain, Vγ9Vδ2 TCR triggering involves the V-domain of BTN3A2/BTN3A3. Moreover, PAg/B30.2 interaction, and the critical γδ-T-cell-activating V-domain, localize to different molecules. Secondly, this distinct topology as well as intracellular trafficking and conformation of BTN3A heteromers or ancestral-like BTN3A homomers are controlled by molecular interactions of the BTN3 juxtamembrane region. Finally, the ability of PAg not simply to bind BTN3A-B30.2, but to promote its subsequent interaction with the BTN2A1-B30.2 domain, is essential for T-cell activation. Defining these determinants of cooperation and division of labor in BTN proteins deepens understanding of PAg sensing and elucidates a mode of action potentially applicable to other BTN/BTNL family members.

Human γδ T cell sensing of AMPK-dependent metabolic tumor reprogramming through TCR recognition of EphA2.

Human γδ T cells contribute to tissue homeostasis and participate in epithelial stress surveillance through mechanisms that are not well understood. Here, we identified ephrin type-A receptor 2 (EphA2) as a stress antigen recognized by a human Vγ9Vδ1 TCR. EphA2 is recognized coordinately by ephrin A to enable γδ TCR activation. We identified a putative TCR binding site on the ligand-binding domain of EphA2 that was distinct from the ephrin A binding site. Expression of EphA2 was up-regulated upon AMP-activated protein kinase (AMPK)-dependent metabolic reprogramming of cancer cells, and coexpression of EphA2 and active AMPK in tumors was associated with higher CD3 T cell infiltration in human colorectal cancer tissue. These results highlight the potential of the human γδ TCR to cooperate with a co-receptor to recognize non-MHC-encoded proteins as signals of cellular dysregulation, potentially allowing γδ T cells to sense metabolic energy changes associated with either viral infection or cancer.

Aryloxy Diester Phosphonamidate Prodrugs of Phosphoantigens (ProPAgens) as Potent Activators of Vγ9/Vδ2 T-Cell Immune Responses.

Vγ9/Vδ2 T-cells are activated by pyrophosphate-containing small molecules known as phosphoantigens (PAgs). The presence of the pyrophosphate group in these PAgs has limited their drug-like properties because of its instability and polar nature. In this work, we report a novel and short Grubbs olefin metathesis-mediated synthesis of methylene and difluoromethylene monophosphonate derivatives of the PAg (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBP) as well as their aryloxy diester phosphonamidate prodrugs, termed ProPAgens. These prodrugs showed excellent stability in human serum (t1/2 > 12 h) and potent activation of Vγ9/Vδ2 T-cells (EC50 ranging from 5 fM to 73 nM), which translated into sub-nanomolar γδ T-cell-mediated eradication of bladder cancer cells in vitro. Additionally, a combination of in silico and in vitro enzymatic assays demonstrated the metabolism of these phosphonamidates to release the unmasked PAg monophosphonate species. Collectively, this work establishes HMBP monophosphonate ProPAgens as ideal candidates for further investigation as novel cancer immunotherapeutic agents.

Framework engineering to produce dominant T cell receptors with enhanced antigen-specific function.

TCR-gene-transfer is an efficient strategy to produce therapeutic T cells of defined antigen specificity. However, there are substantial variations in the cell surface expression levels of human TCRs, which can impair the function of engineered T cells. Here we demonstrate that substitutions of 3 amino acid residues in the framework of the TCR variable domains consistently increase the expression of human TCRs on the surface of engineered T cells.The modified TCRs mediate enhanced T cell proliferation, cytokine production and cytotoxicity, while reducing the peptide concentration required for triggering effector function up to 3000-fold. Adoptive transfer experiments in mice show that modified TCRs control tumor growth more efficiently than wild-type TCRs. Our data indicate that simple variable domain modifications at a distance from the antigen-binding loops lead to increased TCR expression and improved effector function. This finding provides a generic platform to optimize the efficacy of TCR gene therapy in humans.

Development and Validation of a Combined Hypoxia and Immune Prognostic Classifier for Head and Neck Cancer.

PURPOSE: Intratumoral hypoxia and immunity have been correlated with patient outcome in various tumor settings. However, these factors are not currently considered for treatment selection in head and neck cancer (HNC) due to lack of validated biomarkers. Here we sought to develop a hypoxia-immune classifier with potential application in patient prognostication and prediction of response to targeted therapy. EXPERIMENTAL DESIGN: A 54-gene hypoxia-immune signature was constructed on the basis of literature review. Gene expression was analyzed in silico using the The Cancer Genome Atlas (TCGA) HNC dataset (n = 275) and validated using two independent cohorts (n = 130 and 123). IHC was used to investigate the utility of a simplified protein signature. The spatial distribution of hypoxia and immune markers was examined using multiplex immunofluorescence staining. RESULTS: Unsupervised hierarchical clustering of TCGA dataset (development cohort) identified three patient subgroups with distinct hypoxia-immune phenotypes and survival profiles: hypoxialow/immunehigh, hypoxiahigh/immunelow, and mixed, with 5-year overall survival (OS) rates of 71%, 51%, and 49%, respectively (P = 0.0015). The prognostic relevance of the hypoxia-immune gene signature was replicated in two independent validation cohorts. Only PD-L1 and intratumoral CD3 protein expression were associated with improved OS on multivariate analysis. Hypoxialow/immunehigh and hypoxiahigh/immunelow tumors were overrepresented in "inflamed" and "immune-desert" microenvironmental profiles, respectively. Multiplex staining demonstrated an inverse correlation between CA-IX expression and prevalence of intratumoral CD3+ T cells (r = -0.5464; P = 0.0377), further corroborating the transcription-based classification. CONCLUSIONS: We developed and validated a hypoxia-immune prognostic transcriptional classifier, which may have clinical application to guide the use of hypoxia modification and targeted immunotherapies for the treatment of HNC.

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.

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 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.

Synthesis and Biological Evaluation of ( E)-4-Hydroxy-3-methylbut-2-enyl Phosphate (HMBP) Aryloxy Triester Phosphoramidate Prodrugs as Activators of Vγ9/Vδ2 T-Cell Immune Responses.

The aryloxy triester phosphoramidate prodrug approach has been used with success in drug discovery. Herein, we describe the first application of this prodrug technology to the monophosphate derivative of the phosphoantigen HMBPP and one of its analogues. Some of these prodrugs exhibited specific and potent activation of Vγ9/Vδ2 T-cells, which were then able to lyse bladder cancer cells in vitro. This work highlights the promise of this prodrug technology in the discovery of novel immunotherapeutics.

KRAS Mutation and Consensus Molecular Subtypes 2 and 3 Are Independently Associated with Reduced Immune Infiltration and Reactivity in Colorectal Cancer.

Purpose:KRAS mutation is a common canonical mutation in colorectal cancer, found at differing frequencies in all consensus molecular subtypes (CMS). The independent immunobiological impacts of RAS mutation and CMS are unknown. Thus, we explored the immunobiological effects of KRAS mutation across the CMS spectrum.Experimental Design: Expression analysis of immune genes/signatures was performed using The Cancer Genome Atlas (TCGA) RNA-seq and the KFSYSCC microarray datasets. Multivariate analysis included KRAS status, CMS, tumor location, MSI status, and neoantigen load. Protein expression of STAT1, HLA-class II, and CXCL10 was analyzed by digital IHC.Results: The Th1-centric co-ordinate immune response cluster (CIRC) was significantly, albeit modestly, reduced in KRAS-mutant colorectal cancer in both datasets. Cytotoxic T cells, neutrophils, and the IFNγ pathway were suppressed in KRAS-mutant samples. The expressions of STAT1 and CXCL10 were reduced at the mRNA and protein levels. In multivariate analysis, KRAS mutation, CMS2, and CMS3 were independently predictive of reduced CIRC expression. Immune response was heterogeneous across KRAS-mutant colorectal cancer: KRAS-mutant CMS2 samples have the lowest CIRC expression, reduced expression of the IFNγ pathway, STAT1 and CXCL10, and reduced infiltration of cytotoxic cells and neutrophils relative to CMS1 and CMS4 and to KRAS wild-type CMS2 samples in the TCGA. These trends held in the KFSYSCC dataset.Conclusions:KRAS mutation is associated with suppressed Th1/cytotoxic immunity in colorectal cancer, the extent of the effect being modulated by CMS subtype. These results add a novel immunobiological dimension to the biological heterogeneity of colorectal cancer. Clin Cancer Res; 24(1); 224-33. ©2017 AACR.

The antigenic identity of human class I MHC phosphopeptides is critically dependent upon phosphorylation status.

Dysregulated post-translational modification provides a source of altered self-antigens that can stimulate immune responses in autoimmunity, inflammation, and cancer. In recent years, phosphorylated peptides have emerged as a group of tumour-associated antigens presented by MHC molecules and recognised by T cells, and represent promising candidates for cancer immunotherapy. However, the impact of phosphorylation on the antigenic identity of phosphopeptide epitopes is unclear. Here we examined this by determining structures of MHC-bound phosphopeptides bearing canonical position 4-phosphorylations in the presence and absence of their phosphate moiety, and examining phosphopeptide recognition by the T cell receptor (TCR). Strikingly, two peptides exhibited major conformational changes upon phosphorylation, involving a similar molecular mechanism, which focussed changes on the central peptide region most critical for T cell recognition. In contrast, a third epitope displayed little conformational alteration upon phosphorylation. In addition, binding studies demonstrated TCR interaction with an MHC-bound phosphopeptide was both epitope-specific and absolutely dependent upon phosphorylation status. These results highlight the critical influence of phosphorylation on the antigenic identity of naturally processed class I MHC epitopes. In doing so they provide a molecular framework for understanding phosphopeptide-specific immune responses, and have implications for the development of phosphopeptide antigen-specific cancer immunotherapy approaches.

BTN3A1 Discriminates γδ T Cell Phosphoantigens from Nonantigenic 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-Ag's 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.

Endothelial protein C receptor is overexpressed in colorectal cancer as a result of amplification and hypomethylation of chromosome 20q.

Endothelial Protein C Receptor (EPCR) is a Major Histocompatibility Complex homologue, with established roles downregulating coagulation and in endothelial protection. Expressed predominantly on endothelium, EPCR affects inflammatory, apoptotic and cell proliferation pathways by binding to activated protein C (APC). However, EPCR can also be expressed on cancer cells, although the underlying reasons are unclear. Moreover, although EPCR has been linked with chemosensitivity in lung cancer, its clinical significance in many tumours is unknown. Here, we explored its significance in colorectal cancer (CRC). Bioinformatic methods revealed EPCR overexpression in many epithelial cancers, which was confirmed on CRC epithelial tumour cells by immunohistochemistry. EPCR upregulation resulted from gene amplification and DNA hypomethylation, and occurred in concert with a cohort of neighbouring genes on chromosome 20q, a region previously implicated in chemoresistance. As in endothelial cells, EPCR reproducibly mediated ERK pathway activation in a model CRC cell line following APC treatment. However, EPCR knockdown studies failed to highlight compelling EPCR-intrinsic impact on CRC cell phenotype, with limited effects on chemosensitivity and no effect on invasion observed, while EPCR appeared to decrease CRC cell migration. Consistent with these observations, differential EPCR expression did not influence response to chemotherapy in a human CRC cohort. Our results provide a compelling explanation for how EPCR is upregulated in diverse epithelial malignancies. They indicate that the clinical significance of EPCR varies across different tumour types. Furthermore, they raise the possibility that the prognostic significance of EPCR in certain tumours relates significantly to co-upregulation of neighbouring genes on chromosome 20q. Therefore, efforts to exploit EPCR as a prognostic marker should be focussed on specific tumours, and in such scenarios EPCR-co-dysregulated genes may represent potential axes for therapeutic intervention.

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

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.

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.