Surface plasmon resonance (SPR) analysis

Transcription

1 Surface plasmon resonance (SPR) analysis Soluble CD8αα and was manufactured as described previously. 1,2 The C12C heterodimeric TCR was produced using an engineered disulfide bridge between the constant domains of the TCRα and TCRβ chains, which were expressed separately in BL21 Escherichia coli. Inclusion bodies were resuspended in 8M urea, 20mM Tris-HCl ph 8, 0.5mM Na-EDTA and 1mM DTT. The C12C TCR was refolded by flash dilution in a solution containing 5M urea, 100mM Tris ph8, 2mM Na-EDTA, 400mM L-Arginine-HCl, 0.5mM oxidised glutathione and 5mM reduced glutathione. The refolding solution was then dialysed to eliminate the urea, and the resulting protein solution was purified by gel filtration and HiTrap-Q anion exchange chromatography. Soluble biotinylated pmhci monomers were manufactured as described previously 3. Binding analysis was performed by SPR using a BIAcore 3000 TM equipped with a CM5 sensor chip 4. Biotinylated pmhci was immobilized to streptavidin, which was chemically linked to the chip surface; alternatively, soluble C12C TCR was immobilized by 12H8 mab capture on the chip surface. Solid phase analytes were injected at slow flow rates to ensure uniform distribution on the chip surface. For equilibrium analysis, ten serial dilutions of each fluid phase analyte were carefully prepared in duplicate or triplicate and injected over the relevant sensor chips (experimental and control) at 25 C. All proteins were prepared, purified and concentrated on the day of analysis to reduce the likelihood of aggregation affecting the results. Data were analyzed using BIAevaluation 3.1, Microsoft Excel and Origin 6.1. The equilibrium binding constant (K D ) values were calculated using a nonlinear curve fit (y = (P 1 x)/(p 2 + x)). REFERENCES 1. Wooldridge L, van den Berg HA, Glick M, et al. Interaction between the CD8 coreceptor and major histocompatibility complex class I stabilizes T cell receptor-antigen complexes at the cell surface. J Biol Chem. 2005;280(30): Gao GF, Gerth UC, Wyer JR, et al. Assembly and crystallization of the complex between the human T cell coreceptor CD8alpha homodimer and HLA-A2. Protein Sci. 1998;7(5): Price DA, Brenchley JM, Ruff LE, et al. Avidity for antigen shapes clonal dominance in CD8+ T cell populations specific for persistent DNA viruses. J Exp Med. 2005;202(10):

2 4. Wyer JR, Willcox BE, Gao GF, et al. T cell receptor and coreceptor CD8 alphaalpha bind peptide-mhc independently and with distinct kinetics. Immunity. 1999;10(2):

3 Figure S1. Surface plasmon resonance analyses of soluble CD8αα and cognate TCR binding to HLA-B*2705-wt and HLA-B*2705-D227K/T228A proteins refolded with the KK10 peptide A. SPR equilibrium binding of soluble CD8αα to HLA-A*0201-wt, HLA-B*2705-wt, and HLA- B*2705-D227K/T228A. The mean response for each concentration is plotted (n = 3). B. SPR equilibrium binding of a soluble KK10-specific TCR (C12C) to HLA-B*2705-wt and HLA- B*2705-D227K/T228A. The mean response for each concentration is plotted (n = 2). The equilibrium dissociation constant (K D ) values were calculated assuming 1:1 Langmuir binding and plotted using a nonlinear curve fit (y = (P 1 x)/(p 2 + x)). Experimental details are provided in Supplementary Materials and Methods. Figure S2. Genetic coding of KK10-specific TCRβ clonotypes Shown are the observed nucleotide sequences coding for the TRBV4-3/TRBJ1-3 clonotypes, together with the contributions from the TRBV (green), TRBD (orange), and TRBJ (blue) genes that would require the minimum number of nucleotide additions (black). Potential P-additions are underlined. The conserved amino acids in the CASSXGXXXNTIY motif are predominantly germline-encoded. The variable amino acid at position 5 (counting from C) was either a fully germline-encoded glutamine (in 5 of the 15 TRBV4-3/TRBJ1-3 clonotypes) or a proline, the

4 germline-encoding of which was largely dependent on P-additions from the 5 end of the TRBD genes. In many of the TRBV4-3/TRBJ1-3 clonotypes, the variable amino acid at position 7 could be either fully or partially TRBD gene-encoded. The multiple lines per nucleotide sequence display the multiple potential recombination events, i.e. different gene contributions and nucleotide additions, that could produce the observed nucleotide sequences with the minimum number of nucleotide additions. Figure S3. TRBV4-3/TRBJ1-3 clones have high antigen sensitivity levels for wt KK10 (A) wt KK10 sensitivity (EC50 for Cr 51 release) of KK10-specific CD8 + T-cell clones grouped according to TRBV/TRBJ usage. Each symbol represents one clone. (B) Similar results are shown with a threshold limit of antigen sensitivity set at 10-8 M to exclude poorly sensitive clones. Statistical analyses were conducted using the Mann-Whitney U-test with Bonferroni correction for multiple comparisons (P< was considered significant). ** and *** indicate P values <0.01 and <0.001, respectively. Figure S4. Activation of KK10-specific CD8 + T-cell clones by wt or Nef viruses Polyfunctional profiles are shown for one high and one low avidity KK10-specific CD8 + T-cell clone after incubation for 6 hours with primary HLA-B CD4 + T-cells presenting similar levels of p24 expression (i.e. 45% or 46%) at day 3 post-infection with wt or Nef viruses (E:T ratio 10:1).

5 Figure S5. Reactivity of KK10-specific CD8 + T-cell clones for wt or L 268 M peptides EC50 values for HLA B*2705-restricted KK10-specific CD8 + T-cell clones (n=11) were determined in standard chromium release assays using peptide (wt or L268M) titrations and a common antigen presenting HLA-B* B-cell line. Ten clones (C2A, E3A, G11C, G12C, E2C, F9A, G10A, G10H, H8B, H9B) displayed lower AgS levels in response to L 268 M compared to wt KK10; only clone C12C showed the inverse pattern.

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