Fig 2: Exogenous AA promotes CD39+?d Tregs polarization in vitro.(A–D) Exogenous AA (5 µM) induced the expression of CD39 on ?d T cells. ?d T cells sorted from BALB/c mice were treated with AA (5 µM) in vitro, and the expression of CD39 on ?d T cells was determined by flow cytometry at 12 hours (A) and 36 hours (C). The percentages of CD39 positive cells at 12 hours (B) and 36 hours (D) were calculated and compared using an unpaired t test (n = 3). (E) Exogenous AA (5 µM) decreased the production of IFN-? in ?d T cells. The percentages of IFN-?+ cells by intracellular staining at 36 hours (F) were calculated and compared using an unpaired t test (n = 3). (G) AA-induced CD39+?d Tregs inhibited the proliferation of CD8+cells. AA-induced CD39+?d Tregs sorted by flow cytometry were then mix with CFSE-labeled aß T cells from BALB/c mice at a 1:5 ratio and activated by anti-CD3/CD28 mAb with or without anti-CD39 mAb/A2AR inhibitor AZD4635. CFSE was determined by flow cytometry, gating on CD8+cells. (H) Percentage of CFSE-low cells were calculated and compared using 1-way ANOVA followed by Tukey’s multiple-comparison test (n = 3). Data are represented as mean ± SEM of 2–3 independent experiments.

Fig 3: Signaling capacity, expression, and isolation of stoichiometrically defined TCR-CD3/pMHC complexes, related to STAR Methods(A) Calcium signaling on SLBs. Jurkat T cells, with endogenous TCR genes inactivated using CRISPR-Cas9, were transfected with cDNA encoding the aß subunits of the tumor-reactive, affinity-matured GPa3b17 TCR (KD = 13 pM; Liddy et al., 2012). The 1G4 TCR binds to the 9V variant of the NY-ESO antigen complexed with HLA-A2 with an affinity (KD = 7.2 µM) more typical of pMHC/TCR interactions (Chen et al., 2005). Cell lines were generated that expressed the TCR at wild-type (WT) levels, i.e., comparable to Jurkat cells (GPa3b17: 11,262 ± 1,532 TCRs/cell; 1G4: 17,141 ± 5,616 TCRs/cell) and low (Lo) levels (GPa3b17: 236 ± 181 TCRs/cell; 1G4: 488 ± 252 TCRs/cell). The cells were allowed to form contacts with nickelated SLBs presenting gp100/HLA-A2 or 9V/HLA-A2 monomers. Calcium responses were monitored using Fluo-4 AM fluorescence as a readout. Inactivation of ZAP70 and the TCR using CRISPR-Cas9 confirmed that the responses were TCR dependent. Data are presented as means ± SD, n = 3–4 biological replicates; the example shown is representative of two experiments.(B) Tests of CD69 upregulation induced by GPa3b17 TCR signaling. Jurkat T cells expressing wild-type amounts of GPa3b17 TCRs were incubated overnight with wild-type THP-1 cells and THP-1 cells whose expression of ß2-microglobulin was prevented using CRISPR-Cas9, in the presence and absence of 100 µM gp100 peptide. CD69 expression was analyzed by flow cytometry. Data are presented as means ± SEM, n = 3 biological replicates; the example shown is representative of two experiments. Notably, while signaling was peptide- and pMHC-dependent (p < 0.05, Student’s t test), the GPa3b17 TCR was also triggered in the absence of the gp100 peptide.(C) Schematic showing the design of the expression constructs. DNA sequences encoding GPa3b17 TCR-aß and CD3 proteins including a GFP2-tagged CD3-d chain were cloned into three lentiviral expression vectors used to stably transduce CHO cells. The lentiviral constructs, named after the subunits they encoded, were called a?e?, ße?, and d-GFP2. Where necessary, viral 2A sequences (blue arrows) were used to allow expression of multiple proteins by a single virus. Kozak sequences and stop codons are indicated by green and red circles.(D) SDS-PAGE analysis of purified gp100/HLA-A2/TCR, gp100/HLA-A2/TCR/UCHT1 (1/1/2 equiv), and UCHT1 Fab (2 equiv) under reducing (R, left) and non-reducing conditions (NR, right).(E) Binding of UCHT1 Fab to gp100/HLA-A2/TCR assessed by blue native PAGE. The gp100/HLA-A2/TCR was mixed with UCHT1 Fab at the indicated molar ratios prior to loading on the gel.(F) SEC analysis of the TCR in the absence and presence of UCHT1 Fab (3 equiv). For reference, the black trace shows the SEC trace for 1 equiv of UCHT1 Fab alone.(G) Titration of gp100/HLA-A2/TCR and UCHT1 Fab monitored by SEC, demonstrating that two UCHT1 Fab molecules can be bound per gp100/HLA-A2/TCR complex.(H) Flow cytometric analyses of GFP fluorescence (left), UCHT1-Phycoerythrin (PE, middle), and Alexa Fluor 647-labeled gp100/HLA-A2 (pMHC, right) staining of untransduced CHO cells (black), and CHO cells transduced with the following lentivirus combinations: d-GFP2/a?e?/ße? (green), d-GFP2/a?e/ße? (orange), and d-GFP2/a?e/ße (cyan). The median fluorescence intensities following pMHC staining were 4,555 (no virus), 134,852 (d-GFP2/a?e?/ße?), 143,808 (d-GFP2/a?e/ße?), and 115,801 (d-GFP2/a?e/ße). Expression of a gp100/HLA-A2 binding form of the GPa3b17 TCR was not strictly reliant on CD3-?.

Fig 4: Transmembrane assembly including the contribution of sterol lipid(A) Extracellular view of the transmembrane domain (layer 3).(B) The two CD3-? chains (shown in white) are positioned in very different environments within the CD3 TM assembly. The view is related to Figure 4A by a 165° rotation around the longest axis of the complex. Residues interacting with CD3-? (blue) and CD3-?' (cyan) were identified using the program PISA (Krissinel and Henrick, 2007) and are shown as sticks and with their atomic van der Waals radii as transparent spheres.(C) Tight associations in the outer leaflet portions of the TCR-a and TCR-ß TMs with CD3-ed and CD3-e'?, respectively, enable the intra-membrane neutralization of positive charges in TCR-a and TCR-ß. Hydrogen bonds and salt bridges in (A) and (C) are depicted by black dashed lines.(D) Extra non-protein density (transparent blue surface) was observed in the outer leaflet between the TM helices of CD3-?' and CD3-?, likely matching a cholesterol molecule.

Fig 5: Stabilization of the TCR-aß and CD3 heterodimer linker regions by the connecting peptides(A) The linker region of the TCR-aß heterodimer. The TCR-a CP helix fills the space underneath the Cß domain, stabilizing the linker region. The close-up view shows the intermolecular disulfide bridge (yellow), securing the position of the CP helix. The cystine is represented as sticks, with the atomic van der Waals radii as transparent spheres.(B and C) The rigidity of the CD3-e'? (B) and CD3-ed (C) heterodimers is reinforced by the tight packing of their connecting peptide regions, and most notably by a pair of interacting cystines. The close-up views show the cystines as sticks, with their atomic van der Waals radii as transparent spheres. Chemically identical subunits at different positions in the TCR assembly are distinguished by the prime symbol.(D) Superimposing CD3-e'? and CD3-ed on CD3-e' and CD3-e reveals the remarkably similar architecture of the two heterodimeric CD3 assemblies as putative docking modules.(E) The marked tilt in the ectodomain of the TCR-aß heterodimer imposed by the linker region, for comparison with similarly tilted ectodomains of the CD3-e'? and CD3-ed heterodimers in (D). The topologies of the heterodimers likely facilitate ectodomain docking and the simultaneous close association of their TM regions.