il 7 receptor α  (Sino Biological)

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: In general, non-receptor interacting loops were deleted from the WT-IL7 sequence and loops connecting the adjacent helices were modeled using Rosetta Loop Remodeler and Rosetta fix backbone design function. The sequence of the designed model was extracted and submitted to AlphaFold (monomer and multimer mode for structure and protein-receptor binding prediction respectively) as a preliminary validation of the Rosetta-remodeled protein. Iterations of the bad models (models that do not fold into the expected structure or models that did not predict to bind to the receptors) back to the design stage were performed. Models that passed the AlphaFold validation proceeded to subsequent in vitro assay using yeast display system and flow cytometry to determine their relative binding affinity to IL-7 receptors in comparison to WT-IL7.

Article Snippet: Receptor binding was done by incubation of yeast with a final concentration of 50 nM IL-7 receptor-α (IL7Rα; Sino Biological Cat: 50090-M08H) consisting of a C-terminal polyhistidine tag for 30 min at 4°C on a mini shaker agitated at 600 rpm.

Techniques: Sequencing, Binding Assay, Biomarker Discovery, In Vitro, Flow Cytometry, Comparison

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: Blueprint of the WT-IL7 was shown on the left of the figure. The connectivity of the functioning helixes was connected in a manner that requires extremely long protein loops by design (i.e. helices were not connected to the closest adjacent helixes but to the opposite helix). Loops that were not interacted with the IL-7 receptors were deleted and the helixes were reconnected in a clockwise manner via new protein linkers connecting to the adjacent helixes. The blueprint of the redesigned protein was shown at the right side of the figure. Protein structures are colored as rainbow (from N-to-C terminus with the order of Blue-Green-Yellow-Red).

Article Snippet: Receptor binding was done by incubation of yeast with a final concentration of 50 nM IL-7 receptor-α (IL7Rα; Sino Biological Cat: 50090-M08H) consisting of a C-terminal polyhistidine tag for 30 min at 4°C on a mini shaker agitated at 600 rpm.

Techniques:

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: ( A ) AlphaFold validation of the first loop design version of Neo-7 (Neo-7 LDv1) using the default (left) and single sequence mode (right). ( B ) AlphaFold validation of the second loop design version of Neo-7 (left; Neo-7 LDv2) and Neo-7 LDv2 with mutations (right) favored by Rosetta fix backbone design. ( C ) Crystal structure of human IL-7 in complexation to human IL-7 receptor alpha (PDB ID = 3DI2). ( D ) Superimposition of Neo-7 structures (with or without additional disulfide bridge) predicted by AlphaFold. ( E ) Yeast display and flow cytometry validation of IL-7/Neo-7 bindings towards the IL-7 receptors. The yeast-displayed protein (different redesigned IL-7s) carries a HA-tag while the recombinant IL-7 receptors carry either a HIS tag (IL-7 receptor alpha) or a FC-tag (common-IL-2 family receptor gamma; for detection of IL-2Rγ binding, yeast cells were first incubated with recombinant IL-7Rα, washed, and subsequently incubated with IL-2Rγ.) The signal intensity of the X-axis (conferred by the binding of anti-HA mab) correlates with the expression level of the displayed protein while the signal intensity of the Y-axis (conferred by the binding of the anti-HIS/anti-FC mAb to the recombinant receptors bound to the displayed proteins) correlates with the binding affinity of the displayed proteins towards the IL-7 receptors.

Article Snippet: Receptor binding was done by incubation of yeast with a final concentration of 50 nM IL-7 receptor-α (IL7Rα; Sino Biological Cat: 50090-M08H) consisting of a C-terminal polyhistidine tag for 30 min at 4°C on a mini shaker agitated at 600 rpm.

Techniques: Biomarker Discovery, Sequencing, Flow Cytometry, Recombinant, Binding Assay, Incubation, Expressing

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: ( A ) Inspection of structural and binding interactions of residue mutations Q6P and T45I on Neo-7 towards the murine IL-7R alpha. ( B ) Yeast display and flow cytometry validation of the binding ability of IL-7/Neo-7 variants toward the IL-7 receptors.

Article Snippet: Receptor binding was done by incubation of yeast with a final concentration of 50 nM IL-7 receptor-α (IL7Rα; Sino Biological Cat: 50090-M08H) consisting of a C-terminal polyhistidine tag for 30 min at 4°C on a mini shaker agitated at 600 rpm.

Techniques: Binding Assay, Residue, Flow Cytometry, Biomarker Discovery

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: FPLC profile of E. coli expressed ( A ) WT-IL7 ( B ) refolded WT-IL7 ( C ) Neo-7-Q6P and ( D ) Neo-7-Q6P-T45I. Percentage of purity is calculated from the SEC-FPLC peak profile via Cytiva Unicorn 7 software after affinity chromatography purification. SPR (Biacore) characterization of the binding kinetics of ( E ) Neo-7-Q6P ( F ) Neo-7-Q6P-T45I and ( G ) WT-IL7 towards murine IL-7R alpha. ( H ) 2E8 proliferation assay to investigate the biological activity of the IL-7/Neo-7s expressed by E. coli . Error bars represent standard deviation (n=3).

Article Snippet: Receptor binding was done by incubation of yeast with a final concentration of 50 nM IL-7 receptor-α (IL7Rα; Sino Biological Cat: 50090-M08H) consisting of a C-terminal polyhistidine tag for 30 min at 4°C on a mini shaker agitated at 600 rpm.

Techniques: Software, Affinity Chromatography, Purification, Binding Assay, Proliferation Assay, Activity Assay, Standard Deviation

Journal: PLoS Pathogens

Article Title: Treatment with IL-7 Prevents the Decline of Circulating CD4 + T Cells during the Acute Phase of SIV Infection in Rhesus Macaques

doi: 10.1371/journal.ppat.1002636

Figure Lengend Snippet: (A) Plasma IL-7 concentrations in IL-7-treated and untreated macaques. IL-7-treated animals received 7 weekly injections of 50 µg/kg of body weight of a recombinant, fully glycosylated form of simian IL-7 (the grey shaded area indicates the IL-7-treatment period). The asterisks denote significant differences with baseline values (p<0.05 by paired Student's t test). (B) CD127 expression on total, naïve, memory and effector CD3 + T cells from untreated (blue) and IL-7-treated (red) animals. Average values of mean fluorescence intensity (MFI) ± standard error of the mean (SEM) from each group of macaques are shown. Blue and red asterisks denote significant differences with baseline values (day −7) in untreated and treated animals, respectively (* p<0.05, ** p<0.01, by paired Student's t test).

Article Snippet: Additional mAbs were used to measure the expression of other cell-surface markers, including the IL-7 receptor-α/CD127-PE (clone hIL-7R-M21), CCR5-PE (clone 3A9), CXCR4-PE (clone 12G5) and the activation markers HLA-DR-PE (clone L243/G46-6) and CD25-PE (clone M-A251) (all from BD Biosciences).

Techniques: Recombinant, Expressing, Fluorescence

Journal: PLoS Pathogens

Article Title: Treatment with IL-7 Prevents the Decline of Circulating CD4 + T Cells during the Acute Phase of SIV Infection in Rhesus Macaques

doi: 10.1371/journal.ppat.1002636

Figure Lengend Snippet: Mean levels (± SEM) of SIV plasma viremia (A) and p27 Gag antigenemia (B) in untreated (blue) and IL-7-treated (red) animals. No significant differences were observed between the two groups of animals, with the only exception of SIV plasma viremia on day 4 post-infection, when IL-7-treated animals showed higher levels compared to untreated controls (indicated by the asterisk; p<0.05, by Wilcoxon rank sum test). The grey-shaded area indicates the IL-7-treatment period. (C) Mean number of genome equivalent copies (± SEM) of SIV proviral DNA in mononuclear cells from peripheral blood (days 14 and 77 post-infection) and lymphoid tissues (GALT, days 14–16; axillary lymph nodes, days 25–27 post-infection). No significant differences were observed between the two groups of animals, p>0.05 by Wilcoxon rank sum test.

Article Snippet: Additional mAbs were used to measure the expression of other cell-surface markers, including the IL-7 receptor-α/CD127-PE (clone hIL-7R-M21), CCR5-PE (clone 3A9), CXCR4-PE (clone 12G5) and the activation markers HLA-DR-PE (clone L243/G46-6) and CD25-PE (clone M-A251) (all from BD Biosciences).

Techniques: Infection

Journal: PLoS Pathogens

Article Title: Treatment with IL-7 Prevents the Decline of Circulating CD4 + T Cells during the Acute Phase of SIV Infection in Rhesus Macaques

doi: 10.1371/journal.ppat.1002636

Figure Lengend Snippet: (A) Mean absolute numbers (± SEM) of circulating total, naïve, memory and effector CD4 + and CD8 + T cells in untreated (blue) and IL-7-treated (red) animals. The naïve (CD28 + 95 − ), memory (CD28 + 95 + ) and effector (CD28 − 95 + ) T-cell subsets were identified using a combination of mAbs against CD28 and CD95. (B) Subset analysis of memory CD4 + and CD8 + T cells in untreated (blue) and IL-7-treated (red) animals. The various memory T-cell subsets (central memory, CM; transitional memory, TM; and effector memory, EM) were identified using a combination of CD28, CD95, CD62L and CD197/CCR7. Absolute counts for each T-lymphocyte subpopulation were calculated by multiplying the percent values obtained by flow cytometry by the total lymphocyte counts obtained from the complete blood counts (CBC). The grey shaded area indicates the IL-7-treatment period; blue and red asterisks denote significant differences versus baseline values in untreated and IL-7-treated animals, respectively (* p<0.05, ** p<0.01, by paired Student's t test).

Article Snippet: Additional mAbs were used to measure the expression of other cell-surface markers, including the IL-7 receptor-α/CD127-PE (clone hIL-7R-M21), CCR5-PE (clone 3A9), CXCR4-PE (clone 12G5) and the activation markers HLA-DR-PE (clone L243/G46-6) and CD25-PE (clone M-A251) (all from BD Biosciences).

Techniques: Flow Cytometry

Journal: PLoS Pathogens

Article Title: Treatment with IL-7 Prevents the Decline of Circulating CD4 + T Cells during the Acute Phase of SIV Infection in Rhesus Macaques

doi: 10.1371/journal.ppat.1002636

Figure Lengend Snippet: (A) Mean levels (± SEM) of cellular proliferation, as measured by expression of the cell-cycling marker Ki67, in CD4 + and CD8 + T cells freshly isolated from untreated (blue) and IL-7-treated (red) animals. (B) Mean levels (± SEM) of spontaneous apoptosis, as measured by Annexin-V binding, in circulating CD4 + and CD8 + T cells from untreated (blue) and IL-7-treated (red) animals. (C) Average MFI levels (± SEM) of Bcl-2 expression in circulating CD4 + and CD8 + T cells from untreated (blue) and IL-7-treated (red) animals. The grey shaded area indicates the IL-7-treatment period. The asterisks denote significant differences between untreated and IL-7-treated animals (* p<0.05, ** p<0.01, by Wilcoxon rank sum test).

Article Snippet: Additional mAbs were used to measure the expression of other cell-surface markers, including the IL-7 receptor-α/CD127-PE (clone hIL-7R-M21), CCR5-PE (clone 3A9), CXCR4-PE (clone 12G5) and the activation markers HLA-DR-PE (clone L243/G46-6) and CD25-PE (clone M-A251) (all from BD Biosciences).

Techniques: Expressing, Marker, Isolation, Binding Assay

Journal: PLoS Pathogens

Article Title: Treatment with IL-7 Prevents the Decline of Circulating CD4 + T Cells during the Acute Phase of SIV Infection in Rhesus Macaques

doi: 10.1371/journal.ppat.1002636

Figure Lengend Snippet: Lymph node (upper panels) and gut (lower panels) biopsies were obtained from all animals on days 25–27 and on days 14–16 post-infection, respectively. (A) Mean levels of spontaneous apoptosis, as measured by Annexin-V binding, in total, naïve, memory and effector CD4 + and CD8 + T cells isolated from axillary lymph node and terminal ileum biopsies from untreated (blue) and IL-7-treated (red) animals. The differences between untreated and IL-7-treated animals were analyzed by unpaired t-test (similar results were obtained by Wilcoxon rank sum test). (B) Mean levels of spontaneous apoptosis, as measured by Annexin-V binding, on total, naïve, memory and effector CD4 + and CD8 + T cells freshly isolated from axillary lymph node and terminal ileum biopsies from untreated (blue) and IL-7-treated (red) animals, after the exclusion of 4 rapid progressor (RP) animals. The differences between untreated and IL-7-treated animals were analyzed by unpaired t-test.

Article Snippet: Additional mAbs were used to measure the expression of other cell-surface markers, including the IL-7 receptor-α/CD127-PE (clone hIL-7R-M21), CCR5-PE (clone 3A9), CXCR4-PE (clone 12G5) and the activation markers HLA-DR-PE (clone L243/G46-6) and CD25-PE (clone M-A251) (all from BD Biosciences).

Techniques: Infection, Binding Assay, Isolation

Journal: PLoS Pathogens

Article Title: Treatment with IL-7 Prevents the Decline of Circulating CD4 + T Cells during the Acute Phase of SIV Infection in Rhesus Macaques

doi: 10.1371/journal.ppat.1002636

Figure Lengend Snippet: Mean absolute numbers (± SEM) of total CD4 + and CD8 + T cells responding to overlapping peptides derived from the Tat and Gag proteins of SIV in untreated (blue) and IL-7-treated (red) animals. The asterisk indicates a significant difference between total responses in IL-7-treated vs. untreated macaques.

Article Snippet: Additional mAbs were used to measure the expression of other cell-surface markers, including the IL-7 receptor-α/CD127-PE (clone hIL-7R-M21), CCR5-PE (clone 3A9), CXCR4-PE (clone 12G5) and the activation markers HLA-DR-PE (clone L243/G46-6) and CD25-PE (clone M-A251) (all from BD Biosciences).

Techniques: Derivative Assay

Journal: PLoS Pathogens

Article Title: Treatment with IL-7 Prevents the Decline of Circulating CD4 + T Cells during the Acute Phase of SIV Infection in Rhesus Macaques

doi: 10.1371/journal.ppat.1002636

Figure Lengend Snippet: (A) Mean absolute numbers (± SEM) of CD4 + and CD8 + T cells producing one cytokine (single-producer, SP), two cytokines (double-producer, DP) or all three cytokines (triple-producer, TP) in response to SIV Tat peptide stimulation in untreated (shades of blue) and IL-7-treated (shades of red) animals. The asterisks indicate significant differences between SP T cells in untreated vs. IL-7-treated animals, as analyzed by Wilcoxon rank sum test. (B) Mean absolute numbers of SIV Tat-responding CD4 + and CD8 + T cells in untreated (U) and IL-7-treated (IL-7) animals. The bars indicate the mean numbers of total responding cells; the colors indicate the mean numbers of IFN-γ, IL-2, or MIP-1β SP cells (shades of blue), IFN-γ/IL-2, IFN-γ/MIP-1β or IL-2/MIP-1β DP cells (shades of green), and IFN-γ/IL-2/MIP-1β TP cells (purple). The numbers above each bar indicate the fraction of monkeys that gave a measurable response over background to SIV Tat peptides at the corresponding time point. The grey shaded areas indicate the IL-7-treatment period. The asterisk indicates a significant difference between total responses in IL-7-treated vs, untreated macaques. (C) Qualitative analysis of SIV Tat-specific CD8 + T-cell responses in IL-7-treated and untreated macaques on day 62 post-infection. The pie charts indicate the average contribution of the various functional subpopulations of Tat-responding CD8 + T cells (single-, double- and triple-producing, SP, DP, TP, respectively) to the total number of Tat-responding cells in untreated and IL-7-treated animals at day 62 post-infection. The p value was calculated by permutation analysis using the Spice software.

Article Snippet: Additional mAbs were used to measure the expression of other cell-surface markers, including the IL-7 receptor-α/CD127-PE (clone hIL-7R-M21), CCR5-PE (clone 3A9), CXCR4-PE (clone 12G5) and the activation markers HLA-DR-PE (clone L243/G46-6) and CD25-PE (clone M-A251) (all from BD Biosciences).

Techniques: Infection, Functional Assay, Software