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.

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.

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.

Phosphoantigen sensing combines TCR-dependent recognition of the BTN3A IgV domain and germline interaction with BTN2A1.

Vγ9Vδ2 T cells play critical roles in microbial immunity by detecting target cells exposed to pathogen-derived phosphoantigens (P-Ags). Target cell expression of BTN3A1, the "P-Ag sensor," and BTN2A1, a direct ligand for T cell receptor (TCR) Vγ9, is essential for this process; however, the molecular mechanisms involved are unclear. Here, we characterize BTN2A1 interactions with Vγ9Vδ2 TCR and BTN3A1. Nuclear magnetic resonance (NMR), modeling, and mutagenesis establish a BTN2A1-immunoglobulin V (IgV)/BTN3A1-IgV structural model compatible with their cell-surface association in cis. However, TCR and BTN3A1-IgV binding to BTN2A1-IgV is mutually exclusive, owing to binding site proximity and overlap. Moreover, mutagenesis indicates that the BTN2A1-IgV/BTN3A1-IgV interaction is non-essential for recognition but instead identifies a molecular surface on BTN3A1-IgV essential to P-Ag sensing. These results establish a critical role for BTN3A-IgV in P-Ag sensing, in mediating direct or indirect interactions with the γδ-TCR. They support a composite-ligand model whereby intracellular P-Ag detection coordinates weak extracellular germline TCR/BTN2A1 and clonotypically influenced TCR/BTN3A-mediated interactions to initiate Vγ9Vδ2 TCR triggering.

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.

Transcriptional profiling of human Vδ1 T cells reveals a pathogen-driven adaptive differentiation program.

γδ T cells are generally considered innate-like lymphocytes, however, an "adaptive-like" γδ compartment has now emerged. To understand transcriptional regulation of adaptive γδ T cell immunobiology, we combined single-cell transcriptomics, T cell receptor (TCR)-clonotype assignment, ATAC-seq, and immunophenotyping. We show that adult Vδ1+ T cells segregate into TCF7+LEF1+Granzyme Bneg (Tnaive) or T-bet+Eomes+BLIMP-1+Granzyme B+ (Teffector) transcriptional subtypes, with clonotypically expanded TCRs detected exclusively in Teffector cells. Transcriptional reprogramming mirrors changes within CD8+ αß T cells following antigen-specific maturation and involves chromatin remodeling, enhancing cytokine production and cytotoxicity. Consistent with this, in vitro TCR engagement induces comparable BLIMP-1, Eomes, and T-bet expression in naive Vδ1+ and CD8+ T cells. Finally, both human cytomegalovirus and Plasmodium falciparum infection in vivo drive adaptive Vδ1 T cell differentiation from Tnaive to Teffector transcriptional status, alongside clonotypic expansion. Contrastingly, semi-invariant Vγ9+Vδ2+ T cells exhibit a distinct "innate-effector" transcriptional program established by early childhood. In summary, adaptive-like γδ subsets undergo a pathogen-driven differentiation process analogous to conventional CD8+ T cells.

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.

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.

Generation of Stable Isopentenyl Monophosphate Aryloxy Triester Phosphoramidates as Activators of Vγ9Vδ2 T Cells.

Aryloxy triester phosphoramidate prodrugs of the monophosphate derivatives of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) were synthesized as lipophilic derivatives that can improve cell uptake. Despite the structural similarity of IPP and DMAPP, it was noted that their phosphoramidate prodrugs exhibited distinct stability profiles in aqueous environments, which we show is due to the position of the allyl bond in the backbones of the IPP and DMAPP monophosphates. As the IPP monophosphate aryloxy triester phosphoramidates showed favorable stability, they were subsequently investigated for their ability to activate Vγ9/Vδ2 T cells and they showed promising activation of this subset of T cells. Together, these findings represent the first report of IPP and DMAPP monophosphate prodrugs and the ability of IPP aryloxy triester phosphoramidate prodrugs to activate Vγ9/Vδ2 T cells highlighting their potential as possible immunotherapeutics.

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.

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.

Detection of antibodies to the SARS-CoV-2 spike glycoprotein in both serum and saliva enhances detection of infection.

BACKGROUND: 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 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. METHODS: We systemically developed an ELISA assay, optimising different antigens and amplification steps, in serum and saliva from symptomatic and asymptomatic SARS-CoV-2-infected subjects. RESULTS: 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, but not nucleocapsid, IgG, IgA and IgM antibody responses were readily detectable in saliva from non-hospitalized symptomatic and asymptomatic individuals. Antibody responses in saliva and serum were largely independent of each other and symptom reporting. CONCLUSIONS: Detecting antibody responses in both saliva and serum is optimal for determining virus exposure and understanding immune responses after SARS-CoV-2 infection. FUNDING: This work was funded by the University of Birmingham, the National Institute for Health Research (UK), the NIH National Institute for Allergy and Infectious Diseases, the Bill and Melinda Gates Foundation and the University of Southampton.

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.

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.