- Email: [email protected]
Docking Complete: A Step Further toward the Holy Grail of γδ T Cell Biology
Immunity
Previews Docking Complete: A Step Further toward the Holy Grail of gd T Cell Biology Ningning Cai,1,2 Shuai Han,1,2 and Yonghui Zhang1,* 1School of Pharmaceutical Sciences; Beijing Advanced Innovation Center for Structural Biology, MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, 100084 Beijing, China 2These authors contributed equally *Correspondence: [email protected] https://doi.org/10.1016/j.immuni.2019.10.013
The direct ligands recognized by gd T cell receptors (gd TCRs) remain uncertain and controversial. In a study appearing in this issue, Willcox et al. use surface plasmon resonance and isothermal titration calorimetry to demonstrate that B7-like molecule BTNL3 makes physical contact with V4g-bearing TCRs on gd T cells. Antigens (mainly peptides) lodged in the grooves of major histocompatibility complex (MHC) are anchored to ab T cell receptors (TCRs) during antigen recognition, which is the core procedure of cellular immunity dominated by ab T cells. In sharp contrast to this well-understood ‘‘docking process,’’ corresponding paradigm employed by gd T cells, which contribute to both innatelike and adaptive immunity (Vantourout and Hayday, 2013), is blurry. In particular, the direct ligands recognized by gd TCRs still remain largely unknown, forming a central mystery of gd T cell biology. Previous studies have demonstrated how B7-like butyrophilin (BTN) and butyrophilin-like (BTNL) proteins function to modulate the selection and activation of various subsets gd T cells (Arnett and Viney, 2014), and the intracellular region of BTN3A1, which is widely expressed in human cell lines, is now known to be essential for phosphorylated antigen (p-Ags; small, pyrophosphate-containing molecules) binding, thus leading to subsequent Vg9Vd2 T cells activation (Sandstrom et al., 2014; Yang et al., 2019). Vavassori et al. reported direct binding of the BTN3A1 extracellular domain with Vg9Vd2 TCR (Vavassori et al., 2013), but these results have not been reproduced, and there has been considerable ongoing controversy since then about whether or not the extracellular domain of BTN3A1 protein can directly bind with the Vg9Vd2 TCR and serve as a direct ligand. In this issue of Immunity, Willcox et al. (2019) adopted a multifaceted experimental strategy involving surface plasmon resonance (SPR), isothermal
titration calorimetry (ITC), mutagenesis, and computational docking to unequivocally confirm that gd T cell receptors directly interact with BTNL proteins. Fundamentally, their work demonstrates that BTNL3 do indeed mediate gd T cell-mediated immunity, specifically by functioning as direct gd TCR ligands. This is a major breakthrough that validates BTNL3 as direct Vg4 TCR ligand, substantially deepens our understanding of the basic biology of gd T cells, and will almost certainly direct future developments in gd T cell based cancer immunotherapies. There have been hints from previous studies that BTNL3-BTNL8 heterodimers respond to Vg4+ gd TCRs, and multiple mutagenesis experiments have established that BTNL30 s immunoglobulin V (IgV) domain somehow functions in Vg4mediated TCR triggering (Melandri et al., 2018). However, until now, there has been no direct physical evidence demonstrating an interaction between BTNL30 s IgV domain and Vg4 TCRs. Our previous work using atomic force microscopy indicates that the presence of a p-Ag doubles the interaction force between a target cell and a Vg9Vd2 T cell and does so in a BTN3A1-dependent manner (Yang et al., 2019). However, this is not direct evidence of a Vg9Vd2 TCR and BTN3A1 interaction. The new Willcox study uses two robustly quantitative analytical techniques common in biophysics research to unambiguously demonstrate binding of BTNL3 IgV and Vg4 TCR. First, SPR was utilized to detect the direct binding of recombinant BTNL3 and BTNL8 IgV proteins with a range of soluble Vg TCRs (Vg2, Vg3, Vg4). SPR
can quantitatively detect real-time and dynamic binding between diverse biomolecules including DNA, proteins, and oligosaccharides. Strong signals for BTNL3 IgV with the Vg4 TCR, but not with the Vg2 or Vg3 TCRs, established the specific binding between BTNL3 and Vg4 TCR; notably, the BTNL8 IgV domain alone did not bind to any tested TCRs. Further equilibrium binding measurements by SPR determined that the BTNL3Vg4 TCR binding affinity (Kd) was 15–25 mM, a value comparable to many ab TCR-peptide-MHC interactions (0.2–390 mM) (Aleksic et al., 2012). To provide further support for the direct binding between BTNL3 IgV and Vg4 TCR, Willcox et al. (2019) used ITC to validate their results. ITC is also a widely used biophysical analytical method for quantitative determination of binding affinity resulting from binding between two components. Similar to the SPR results, the ITC data also confirmed specific binding between BTNL3 and Vg4 TCR, with a broadly similar affinity of 3.5 mM. Having established the direct binding, Willcox et al. (2019) next generated Vg4 TCR variants with charge reversal mutations for different regions of the CDRg, CDRd, and ‘‘hypervariable region 4’’ (HV4) proteins, which experimentally confirmed that gd TCRs can employ two discrete binding modalities (Figure 1): (1) CDR2g and HV4g-dependent, superantigen-like binding interactions that mediate innate immunity and (2) CDR3dependent, antibody-like interactions that mediate adaptive immunity. Specifically, mutations of CDR2g and HV4g dramatically reduced the binding of BTNL3 with Vg4+ TCR. Further chimeric
Immunity 51, November 19, 2019 ª 2019 Elsevier Inc. 781
Immunity
Previews
Figure 1. Two Distinct Ligand Recognition Modalities of Vg4 TCR and a Possible Model of p-Ag Sensing Interactions of Vg4 T cells with BTNL and EPCR molecules. BTNL3 directly and specifically binds with Vg4 TCR in a superantigen-like binding mode that mediates innate immunity, while EPCR binds with Vg4Vd5 TCR in a CDR3 dependent and antibody-like binding mode that mediates adaptive immunity. CFG face of BTNL3 IgV domain, as well as HV4 and CDR2 regions of Vg4 TCR, are involved in the direct binding of BTNL3 with Vg4 TCR. Interaction of Vg9Vd2-expressing T cells with BTN3A molecules. BTN3A1 and BTN3A2 may form a heterodimer on the cell surface and work together to achieve maximal Vg9Vd2 activation: BTN3A2 first helps with trafficking and cell-surface expression of BTN3A1. P-Ag then binds with BTN3A1 intracellular B30.2 domain and triggers the so-called ‘‘inside-out’’ signal transduction. Subsequent Vg9Vd2 activation is mainly mediated by the BTN3A2 IgV domain, while the BTN3A1 IgV domain may only undergo weak interactions with Vg9Vd2 TCR. Unknown chaperones may also exist during interactions of BTN3A1 or 2 with Vg9Vd2 TCR.
constructs—in which CDR2g and/or the HV4 loop regions of human Vg4 replaced the counterpart regions of human Vg3 and mouse Vg7 (Btnl1.6-responsive) TCRs— gained additional interactivity with BTNL3-BTNL8 heterodimers. In contrast to these superantigen-like binding interactions, the only disruption of binding between endothelial protein C receptor (EPCR, a direct ligand for the human Vg4+TCRV) and Vg4 TCR that Willcox et al. (2019) observed was caused by mutation of CDR3. Going some way to help future efforts seeking to precisely characterize the structural basis of these interactions, their SPR analysis with mutation variants revealed that the CFG face (domain formed by C, F, and G b strands) of BTNL3 mediated the direct interactions with Vg4 TCR. Given this experimental confirmation of two distinct TCR binding modalities, it can now be assumed that gd T cells can collectively provide complementary innate-like and adaptive-like arms to gd T cell responses. Nevertheless, some details of these two ligand interaction modalities remain unclear, so future studies 782 Immunity 51, November 19, 2019
should explore the precise structural interactions, as well as the generality of such bimodal antigen receptor binding in other subsets of gd T cells. Seeking to extend their insights to consideration of potential commonalities between the ligand interaction mode employed by Vg4 and that employed by Vg9, Willcox et al. (2019) undertook computational docking analyses and also generated charge-altering mutations of Vg9Vd2 TCR and BTN3A1 IgV domain residues; these efforts successfully confirmed similar dependence on the HV4 region of Vg9 and CFG face of BTN3A1 IgV domain during Vg9Vd2 T cell activation process. However, no direct physical binding of BTN3A1 to Vg9Vd2 TCR was detected, which raises compelling questions about the unique recognition mechanisms of Vg9Vd2 TCR to BTN3A1. One possibility is that the extracellular domain of BTN3A1 cannot be maintained in a dominant binding conformation in vitro (Yang et al., 2019), making it difficult to detect its binding affinity by ITC or SPR. Moreover, it cannot be excluded that there may be an
unknown chaperone that somehow functions in the interaction between Vg9Vd2 TCR and BTN3A1. Further mechanistic insights about Vg9Vd2 T cell activation were generally revealed based on coexpression of BTN3A1mutant+BTN3A2wt and BTN3A1wt+BTN3A2mutant proteins in Btnl9/ (gene encoding BTN3A) CRA123 cells: CFG mutations of BTN3A1 could be complemented by wild-type BTN3A2, but not vice versa. So BTN3A2’s extracellular IgV domain is required for activation of Vg9Vd2 T cells. Vantourout et al. previously demonstrated that heteromerization of BTN3A2 and BTN3A1 regulates the trafficking and cell-surface expression of BTN3A1 (Vantourout et al., 2018). Since CFG mutated BTN3A2 didn’t affect wild-type BTN3A1’s cell surface expression, the relatively weak Vg9Vd2 activation of BTN3A1wt+BTN3A2mutant probably arose from weak interactions between BTN3A1 IgV domain and Vg9Vd2 TCR. Willcox et al. (2019) pushed the boundary of our understanding further, showing that BTN3A2’s extracellular IgV domain also participated in BTN3A1 mediated Vg9Vd2 activation. These informative and insightful findings have led us to the following possible explanations (Figure 1): perhaps BTN3A1 and BTN3A2 form a heterodimer on target cell membrane. BTN3A1’s intracellular B30.2 domain is known to be responsible for p-Ag binding, while BTN3A2’s extracellular IgV domain is in charge of interacting with Vg9Vd2 TCR to induce subsequent Vg9Vd2 activation. BTN3A1’s extracellular IgV domain may undergo relatively weak interactions with Vg9Vd2 TCR. Thus, BTN3A1 and BTN3A2 co-expression may be needed to achieve maximal Vg9Vd2 activation. Indeed, more detailed work will be needed to address these possibilities—for example, the detection of direct or indirect interactions between BTN3A2 IgV domain and Vg9Vd2 TCR. Chimeras comprising different domains of the BTN3A1 and BTN3A2 proteins can also help to validate these possibilities. There are many other intriguing questions elicited by the fascinating discoveries of this study—for example, whether there are any ‘‘switch molecules’’ like p-Ags that can activate BTNL3 to trigger subsequent Vg4 TCR activation.
Immunity
Previews Importantly, given that BTNL3 binds Vg4 TCR via a superantigen-like binding interaction that mediates innate immunity—which is distinct from Vg4+ T cells’ adaptive, antibody-like modality—what are the physiological implications for human intestinal Vg4+ T cells of the coexistence of these two distinct recognition models? Are these kinds of recognition modalities universal across all gd T cells? Gaps in our understanding remain, but it now seems highly probable that the diligent and dedicated efforts of scientists using approaches including X-ray crystallography and cryoelectron microscopy will soon enable us to directly visualize the long-sought gd TCR-butyrophilin interaction, and this revelation can be expected to drive the rapid development of butyrophilin-targeted therapies. All in all, the Willcox et al. (2019) study demonstrates that docking is complete at the biochemical level and should inform future studies to examine the atomic interactions of the antigen receptor on gd T cells with relevant ligands.
ACKNOWLEDGMENTS
vation of human Vg9Vd2 T cells. Immunity 40, 490–500.
Y.Z. was supported by Beijing Natural Science Foundation (Z190015) and Beijing Advanced Innovation Center for Structural Biology. We thank professor Yan Shi for help in editing.
Vantourout, P., and Hayday, A. (2013). Six-of-thebest: unique contributions of gd T cells to immunology. Nat. Rev. Immunol. 13, 88–100.
REFERENCES Aleksic, M., Liddy, N., Molloy, P.E., Pumphrey, N., Vuidepot, A., Chang, K.M., and Jakobsen, B.K. (2012). Different affinity windows for virus and cancer-specific T-cell receptors: implications for therapeutic strategies. Eur. J. Immunol. 42, 3174–3179. Arnett, H.A., and Viney, J.L. (2014). Immune modulation by butyrophilins. Nat. Rev. Immunol. 14, 559–569. Melandri, D., Zlatareva, I., Chaleil, R.A.G., Dart, R.J., Chancellor, A., Nussbaumer, O., Polyakova, O., Roberts, N.A., Wesch, D., Kabelitz, D., et al. (2018). The gdTCR combines innate immunity with adaptive immunity by utilizing spatially distinct regions for agonist selection and antigen responsiveness. Nat. Immunol. 19, 1352–1365. Sandstrom, A., Peigne´, C.M., Le´ger, A., Crooks, J.E., Konczak, F., Gesnel, M.C., Breathnach, R., Bonneville, M., Scotet, E., and Adams, E.J. (2014). The intracellular B30.2 domain of butyrophilin 3A1 binds phosphoantigens to mediate acti-
Vantourout, P., Laing, A., Woodward, M.J., Zlatareva, I., Apolonia, L., Jones, A.W., Snijders, A.P., Malim, M.H., and Hayday, A.C. (2018). Heteromeric interactions regulate butyrophilin (BTN) and BTN-like molecules governing gd T cell biology. Proc. Natl. Acad. Sci. USA 115, 1039–1044. Vavassori, S., Kumar, A., Wan, G.S., Ramanjaneyulu, G.S., Cavallari, M., El Daker, S., Beddoe, T., Theodossis, A., Williams, N.K., Gostick, E., et al. (2013). Butyrophilin 3A1 binds phosphorylated antigens and stimulates human gd T cells. Nat. Immunol. 14, 908–916. Willcox, C.R., Vantourout, P., Salim, M., Zlatareva, I., Melandri, D., Zanardo, L., George, R., Kjaer, S., Jeeves, M., Mohammed, F., et al. (2019). Butyrophilin-like 3 Directly Binds a Human Vg4+ T Cell Receptor Using a Modality Distinct from Clonally-Restricted Antigen. Immunity 51, this issue, 813–825. Yang, Y., Li, L., Yuan, L., Zhou, X., Duan, J., Xiao, H., Cai, N., Han, S., Ma, X., Liu, W., et al. (2019). A structural change in butyrophilin upon phosphoantigen binding underlies phosphoantigenmediated Vg9Vd2 T cell activation. Immunity 50, 1043–1053.e5.
T Cell Metabolism in a State of Flux Siva Karthik Varanasi,1 Shixin Ma,1 and Susan M. Kaech1,* 1NOMIS Center for Immunobiology and Microbial Pathogenesis, Salk Institute for Biological Studies, 10010 N Torrey Pines Rd, La Jolla, CA 92037, USA *Correspondence: [email protected] https://doi.org/10.1016/j.immuni.2019.10.012
Our knowledge of T cell metabolism relies primarily on studies performed in vitro that may not fully recapitulate physiological conditions in vivo. In this issue of Immunity, Ma et al. find that the in vivo environment dictates the metabolic phenotype of effector CD8+ T cells—particularly their glucose utilization. CD8+ T cells grossly alter their metabolic states following T cell receptor (TCR) activation to accommodate differential energetic and biosynthetic demands as they proliferate and differentiate from naive T (Tn) into effector T (Teff) cells, but our current understanding of this metabolic transition is mostly, if not entirely, based on in vitro cell-culture studies of activated T cells. In this issue of Immunity, Ma et al. (2019) now use in vivo 13C-glucosebased stable isotope labeling (SIL) to trace the metabolism of glucose in CD8+ T cells
activated in vivo during a bacterial infection to those activated in vitro and demonstrate several fundamental differences in glucose utilization between in vivo and in vitro Teff cells. Predominantly, they find that Teff cells generated in vivo display higher rates of mitochondrial respiration and oxidative phosphorylation (OXPHOS) while simultaneously producing lower amounts of lactate compared to those formed in vitro (Figure 1). This study provides one of the first detailed analysis of Teff glucose metabolism in vivo and iden-
tifies important distinctions beween tissue culture and in vivo conditions. Aerobic glycolysis or the so called ‘‘Warburg effect’’ are terms commonly used to describe when cells preferentially increase their rates of glycolysis relative to mitochondrial OXPHOS, even in aerobic conditions, to increase ATP as well as biomass (e.g., nucleotides, amino acids, lipids) (Warburg et al., 1927). The Warburg effect was first described by Dr. Otto Warburg in his pioneering studies in cancer cell metabolism, which was then
Immunity 51, November 19, 2019 ª 2019 Elsevier Inc. 783
Previews Docking Complete: A Step Further toward the Holy Grail of gd T Cell Biology Ningning Cai,1,2 Shuai Han,1,2 and Yonghui Zhang1,* 1School of Pharmaceutical Sciences; Beijing Advanced Innovation Center for Structural Biology, MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, 100084 Beijing, China 2These authors contributed equally *Correspondence: [email protected] https://doi.org/10.1016/j.immuni.2019.10.013
The direct ligands recognized by gd T cell receptors (gd TCRs) remain uncertain and controversial. In a study appearing in this issue, Willcox et al. use surface plasmon resonance and isothermal titration calorimetry to demonstrate that B7-like molecule BTNL3 makes physical contact with V4g-bearing TCRs on gd T cells. Antigens (mainly peptides) lodged in the grooves of major histocompatibility complex (MHC) are anchored to ab T cell receptors (TCRs) during antigen recognition, which is the core procedure of cellular immunity dominated by ab T cells. In sharp contrast to this well-understood ‘‘docking process,’’ corresponding paradigm employed by gd T cells, which contribute to both innatelike and adaptive immunity (Vantourout and Hayday, 2013), is blurry. In particular, the direct ligands recognized by gd TCRs still remain largely unknown, forming a central mystery of gd T cell biology. Previous studies have demonstrated how B7-like butyrophilin (BTN) and butyrophilin-like (BTNL) proteins function to modulate the selection and activation of various subsets gd T cells (Arnett and Viney, 2014), and the intracellular region of BTN3A1, which is widely expressed in human cell lines, is now known to be essential for phosphorylated antigen (p-Ags; small, pyrophosphate-containing molecules) binding, thus leading to subsequent Vg9Vd2 T cells activation (Sandstrom et al., 2014; Yang et al., 2019). Vavassori et al. reported direct binding of the BTN3A1 extracellular domain with Vg9Vd2 TCR (Vavassori et al., 2013), but these results have not been reproduced, and there has been considerable ongoing controversy since then about whether or not the extracellular domain of BTN3A1 protein can directly bind with the Vg9Vd2 TCR and serve as a direct ligand. In this issue of Immunity, Willcox et al. (2019) adopted a multifaceted experimental strategy involving surface plasmon resonance (SPR), isothermal
titration calorimetry (ITC), mutagenesis, and computational docking to unequivocally confirm that gd T cell receptors directly interact with BTNL proteins. Fundamentally, their work demonstrates that BTNL3 do indeed mediate gd T cell-mediated immunity, specifically by functioning as direct gd TCR ligands. This is a major breakthrough that validates BTNL3 as direct Vg4 TCR ligand, substantially deepens our understanding of the basic biology of gd T cells, and will almost certainly direct future developments in gd T cell based cancer immunotherapies. There have been hints from previous studies that BTNL3-BTNL8 heterodimers respond to Vg4+ gd TCRs, and multiple mutagenesis experiments have established that BTNL30 s immunoglobulin V (IgV) domain somehow functions in Vg4mediated TCR triggering (Melandri et al., 2018). However, until now, there has been no direct physical evidence demonstrating an interaction between BTNL30 s IgV domain and Vg4 TCRs. Our previous work using atomic force microscopy indicates that the presence of a p-Ag doubles the interaction force between a target cell and a Vg9Vd2 T cell and does so in a BTN3A1-dependent manner (Yang et al., 2019). However, this is not direct evidence of a Vg9Vd2 TCR and BTN3A1 interaction. The new Willcox study uses two robustly quantitative analytical techniques common in biophysics research to unambiguously demonstrate binding of BTNL3 IgV and Vg4 TCR. First, SPR was utilized to detect the direct binding of recombinant BTNL3 and BTNL8 IgV proteins with a range of soluble Vg TCRs (Vg2, Vg3, Vg4). SPR
can quantitatively detect real-time and dynamic binding between diverse biomolecules including DNA, proteins, and oligosaccharides. Strong signals for BTNL3 IgV with the Vg4 TCR, but not with the Vg2 or Vg3 TCRs, established the specific binding between BTNL3 and Vg4 TCR; notably, the BTNL8 IgV domain alone did not bind to any tested TCRs. Further equilibrium binding measurements by SPR determined that the BTNL3Vg4 TCR binding affinity (Kd) was 15–25 mM, a value comparable to many ab TCR-peptide-MHC interactions (0.2–390 mM) (Aleksic et al., 2012). To provide further support for the direct binding between BTNL3 IgV and Vg4 TCR, Willcox et al. (2019) used ITC to validate their results. ITC is also a widely used biophysical analytical method for quantitative determination of binding affinity resulting from binding between two components. Similar to the SPR results, the ITC data also confirmed specific binding between BTNL3 and Vg4 TCR, with a broadly similar affinity of 3.5 mM. Having established the direct binding, Willcox et al. (2019) next generated Vg4 TCR variants with charge reversal mutations for different regions of the CDRg, CDRd, and ‘‘hypervariable region 4’’ (HV4) proteins, which experimentally confirmed that gd TCRs can employ two discrete binding modalities (Figure 1): (1) CDR2g and HV4g-dependent, superantigen-like binding interactions that mediate innate immunity and (2) CDR3dependent, antibody-like interactions that mediate adaptive immunity. Specifically, mutations of CDR2g and HV4g dramatically reduced the binding of BTNL3 with Vg4+ TCR. Further chimeric
Immunity 51, November 19, 2019 ª 2019 Elsevier Inc. 781
Immunity
Previews
Figure 1. Two Distinct Ligand Recognition Modalities of Vg4 TCR and a Possible Model of p-Ag Sensing Interactions of Vg4 T cells with BTNL and EPCR molecules. BTNL3 directly and specifically binds with Vg4 TCR in a superantigen-like binding mode that mediates innate immunity, while EPCR binds with Vg4Vd5 TCR in a CDR3 dependent and antibody-like binding mode that mediates adaptive immunity. CFG face of BTNL3 IgV domain, as well as HV4 and CDR2 regions of Vg4 TCR, are involved in the direct binding of BTNL3 with Vg4 TCR. Interaction of Vg9Vd2-expressing T cells with BTN3A molecules. BTN3A1 and BTN3A2 may form a heterodimer on the cell surface and work together to achieve maximal Vg9Vd2 activation: BTN3A2 first helps with trafficking and cell-surface expression of BTN3A1. P-Ag then binds with BTN3A1 intracellular B30.2 domain and triggers the so-called ‘‘inside-out’’ signal transduction. Subsequent Vg9Vd2 activation is mainly mediated by the BTN3A2 IgV domain, while the BTN3A1 IgV domain may only undergo weak interactions with Vg9Vd2 TCR. Unknown chaperones may also exist during interactions of BTN3A1 or 2 with Vg9Vd2 TCR.
constructs—in which CDR2g and/or the HV4 loop regions of human Vg4 replaced the counterpart regions of human Vg3 and mouse Vg7 (Btnl1.6-responsive) TCRs— gained additional interactivity with BTNL3-BTNL8 heterodimers. In contrast to these superantigen-like binding interactions, the only disruption of binding between endothelial protein C receptor (EPCR, a direct ligand for the human Vg4+TCRV) and Vg4 TCR that Willcox et al. (2019) observed was caused by mutation of CDR3. Going some way to help future efforts seeking to precisely characterize the structural basis of these interactions, their SPR analysis with mutation variants revealed that the CFG face (domain formed by C, F, and G b strands) of BTNL3 mediated the direct interactions with Vg4 TCR. Given this experimental confirmation of two distinct TCR binding modalities, it can now be assumed that gd T cells can collectively provide complementary innate-like and adaptive-like arms to gd T cell responses. Nevertheless, some details of these two ligand interaction modalities remain unclear, so future studies 782 Immunity 51, November 19, 2019
should explore the precise structural interactions, as well as the generality of such bimodal antigen receptor binding in other subsets of gd T cells. Seeking to extend their insights to consideration of potential commonalities between the ligand interaction mode employed by Vg4 and that employed by Vg9, Willcox et al. (2019) undertook computational docking analyses and also generated charge-altering mutations of Vg9Vd2 TCR and BTN3A1 IgV domain residues; these efforts successfully confirmed similar dependence on the HV4 region of Vg9 and CFG face of BTN3A1 IgV domain during Vg9Vd2 T cell activation process. However, no direct physical binding of BTN3A1 to Vg9Vd2 TCR was detected, which raises compelling questions about the unique recognition mechanisms of Vg9Vd2 TCR to BTN3A1. One possibility is that the extracellular domain of BTN3A1 cannot be maintained in a dominant binding conformation in vitro (Yang et al., 2019), making it difficult to detect its binding affinity by ITC or SPR. Moreover, it cannot be excluded that there may be an
unknown chaperone that somehow functions in the interaction between Vg9Vd2 TCR and BTN3A1. Further mechanistic insights about Vg9Vd2 T cell activation were generally revealed based on coexpression of BTN3A1mutant+BTN3A2wt and BTN3A1wt+BTN3A2mutant proteins in Btnl9/ (gene encoding BTN3A) CRA123 cells: CFG mutations of BTN3A1 could be complemented by wild-type BTN3A2, but not vice versa. So BTN3A2’s extracellular IgV domain is required for activation of Vg9Vd2 T cells. Vantourout et al. previously demonstrated that heteromerization of BTN3A2 and BTN3A1 regulates the trafficking and cell-surface expression of BTN3A1 (Vantourout et al., 2018). Since CFG mutated BTN3A2 didn’t affect wild-type BTN3A1’s cell surface expression, the relatively weak Vg9Vd2 activation of BTN3A1wt+BTN3A2mutant probably arose from weak interactions between BTN3A1 IgV domain and Vg9Vd2 TCR. Willcox et al. (2019) pushed the boundary of our understanding further, showing that BTN3A2’s extracellular IgV domain also participated in BTN3A1 mediated Vg9Vd2 activation. These informative and insightful findings have led us to the following possible explanations (Figure 1): perhaps BTN3A1 and BTN3A2 form a heterodimer on target cell membrane. BTN3A1’s intracellular B30.2 domain is known to be responsible for p-Ag binding, while BTN3A2’s extracellular IgV domain is in charge of interacting with Vg9Vd2 TCR to induce subsequent Vg9Vd2 activation. BTN3A1’s extracellular IgV domain may undergo relatively weak interactions with Vg9Vd2 TCR. Thus, BTN3A1 and BTN3A2 co-expression may be needed to achieve maximal Vg9Vd2 activation. Indeed, more detailed work will be needed to address these possibilities—for example, the detection of direct or indirect interactions between BTN3A2 IgV domain and Vg9Vd2 TCR. Chimeras comprising different domains of the BTN3A1 and BTN3A2 proteins can also help to validate these possibilities. There are many other intriguing questions elicited by the fascinating discoveries of this study—for example, whether there are any ‘‘switch molecules’’ like p-Ags that can activate BTNL3 to trigger subsequent Vg4 TCR activation.
Immunity
Previews Importantly, given that BTNL3 binds Vg4 TCR via a superantigen-like binding interaction that mediates innate immunity—which is distinct from Vg4+ T cells’ adaptive, antibody-like modality—what are the physiological implications for human intestinal Vg4+ T cells of the coexistence of these two distinct recognition models? Are these kinds of recognition modalities universal across all gd T cells? Gaps in our understanding remain, but it now seems highly probable that the diligent and dedicated efforts of scientists using approaches including X-ray crystallography and cryoelectron microscopy will soon enable us to directly visualize the long-sought gd TCR-butyrophilin interaction, and this revelation can be expected to drive the rapid development of butyrophilin-targeted therapies. All in all, the Willcox et al. (2019) study demonstrates that docking is complete at the biochemical level and should inform future studies to examine the atomic interactions of the antigen receptor on gd T cells with relevant ligands.
ACKNOWLEDGMENTS
vation of human Vg9Vd2 T cells. Immunity 40, 490–500.
Y.Z. was supported by Beijing Natural Science Foundation (Z190015) and Beijing Advanced Innovation Center for Structural Biology. We thank professor Yan Shi for help in editing.
Vantourout, P., and Hayday, A. (2013). Six-of-thebest: unique contributions of gd T cells to immunology. Nat. Rev. Immunol. 13, 88–100.
REFERENCES Aleksic, M., Liddy, N., Molloy, P.E., Pumphrey, N., Vuidepot, A., Chang, K.M., and Jakobsen, B.K. (2012). Different affinity windows for virus and cancer-specific T-cell receptors: implications for therapeutic strategies. Eur. J. Immunol. 42, 3174–3179. Arnett, H.A., and Viney, J.L. (2014). Immune modulation by butyrophilins. Nat. Rev. Immunol. 14, 559–569. Melandri, D., Zlatareva, I., Chaleil, R.A.G., Dart, R.J., Chancellor, A., Nussbaumer, O., Polyakova, O., Roberts, N.A., Wesch, D., Kabelitz, D., et al. (2018). The gdTCR combines innate immunity with adaptive immunity by utilizing spatially distinct regions for agonist selection and antigen responsiveness. Nat. Immunol. 19, 1352–1365. Sandstrom, A., Peigne´, C.M., Le´ger, A., Crooks, J.E., Konczak, F., Gesnel, M.C., Breathnach, R., Bonneville, M., Scotet, E., and Adams, E.J. (2014). The intracellular B30.2 domain of butyrophilin 3A1 binds phosphoantigens to mediate acti-
Vantourout, P., Laing, A., Woodward, M.J., Zlatareva, I., Apolonia, L., Jones, A.W., Snijders, A.P., Malim, M.H., and Hayday, A.C. (2018). Heteromeric interactions regulate butyrophilin (BTN) and BTN-like molecules governing gd T cell biology. Proc. Natl. Acad. Sci. USA 115, 1039–1044. Vavassori, S., Kumar, A., Wan, G.S., Ramanjaneyulu, G.S., Cavallari, M., El Daker, S., Beddoe, T., Theodossis, A., Williams, N.K., Gostick, E., et al. (2013). Butyrophilin 3A1 binds phosphorylated antigens and stimulates human gd T cells. Nat. Immunol. 14, 908–916. Willcox, C.R., Vantourout, P., Salim, M., Zlatareva, I., Melandri, D., Zanardo, L., George, R., Kjaer, S., Jeeves, M., Mohammed, F., et al. (2019). Butyrophilin-like 3 Directly Binds a Human Vg4+ T Cell Receptor Using a Modality Distinct from Clonally-Restricted Antigen. Immunity 51, this issue, 813–825. Yang, Y., Li, L., Yuan, L., Zhou, X., Duan, J., Xiao, H., Cai, N., Han, S., Ma, X., Liu, W., et al. (2019). A structural change in butyrophilin upon phosphoantigen binding underlies phosphoantigenmediated Vg9Vd2 T cell activation. Immunity 50, 1043–1053.e5.
T Cell Metabolism in a State of Flux Siva Karthik Varanasi,1 Shixin Ma,1 and Susan M. Kaech1,* 1NOMIS Center for Immunobiology and Microbial Pathogenesis, Salk Institute for Biological Studies, 10010 N Torrey Pines Rd, La Jolla, CA 92037, USA *Correspondence: [email protected] https://doi.org/10.1016/j.immuni.2019.10.012
Our knowledge of T cell metabolism relies primarily on studies performed in vitro that may not fully recapitulate physiological conditions in vivo. In this issue of Immunity, Ma et al. find that the in vivo environment dictates the metabolic phenotype of effector CD8+ T cells—particularly their glucose utilization. CD8+ T cells grossly alter their metabolic states following T cell receptor (TCR) activation to accommodate differential energetic and biosynthetic demands as they proliferate and differentiate from naive T (Tn) into effector T (Teff) cells, but our current understanding of this metabolic transition is mostly, if not entirely, based on in vitro cell-culture studies of activated T cells. In this issue of Immunity, Ma et al. (2019) now use in vivo 13C-glucosebased stable isotope labeling (SIL) to trace the metabolism of glucose in CD8+ T cells
activated in vivo during a bacterial infection to those activated in vitro and demonstrate several fundamental differences in glucose utilization between in vivo and in vitro Teff cells. Predominantly, they find that Teff cells generated in vivo display higher rates of mitochondrial respiration and oxidative phosphorylation (OXPHOS) while simultaneously producing lower amounts of lactate compared to those formed in vitro (Figure 1). This study provides one of the first detailed analysis of Teff glucose metabolism in vivo and iden-
tifies important distinctions beween tissue culture and in vivo conditions. Aerobic glycolysis or the so called ‘‘Warburg effect’’ are terms commonly used to describe when cells preferentially increase their rates of glycolysis relative to mitochondrial OXPHOS, even in aerobic conditions, to increase ATP as well as biomass (e.g., nucleotides, amino acids, lipids) (Warburg et al., 1927). The Warburg effect was first described by Dr. Otto Warburg in his pioneering studies in cancer cell metabolism, which was then
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