anti murine cd8α  (Bio X Cell)

Journal: Thoracic Cancer

Article Title: SOX17 expression in tumor‐penetrating vessels in relation to CD8 + T‐cell infiltration in cancer stroma niches

doi: 10.1111/1759-7714.15464

Figure Lengend Snippet: Representative double immunohistochemical staining using a specific antibody against SOX17 (brown) and CD8 (red) in LUAD tissue specimens. (a) SOX17 immunoreactivity (brown) was observed in cancer cells of five noninvasive lung adenocarcinomas harboring morphological similarity with type II alveolar epithelial cells. Note the strong nuclear staining in cancer cells (upper inset). In contrast, noncancerous type II alveolar epithelial cells indicated by the arrowhead did not show significant SOX17 immunoreactivity (lower inset). (b) Little or no SOX17 immunoreactivity was observed in LUAD cells (indicated by an asterisk) and tumor endothelial cells (arrowhead) in cancer stroma, where a few CD8 + T cells (arrow) were sparsely found. (c–e) Robust SOX17 immunoreactivity was found in tumor endothelial cells (arrowhead) harboring CD8 + T‐cell‐rich cancer stroma. Little SOX17 immunoreactivity was found in LUAD cells (asterisk). (f) Note the intense SOX17 immunoreactivity in tumor endothelial cells around the CD8 + T cells (arrowhead). Scale bar indicates 100 μm in a and 50 μm in (b–e). LUAD, lung adenocarcinoma; SOX17, sex‐determining region Y‐related high‐mobility group box 17 protein.

Article Snippet: Tissue specimens of ovarian high‐grade serous carcinoma were used as positive controls in SOX17 immunohistochemical staining, as they have been reported to be expressed in the same by Shaker et al. Murine monoclonal antibody against CD8α (clone no. C8/144B; cat no. 70306) was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Immunohistochemical staining, Staining

Journal: Molecular Therapy Oncolytics

Article Title: Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research

doi: 10.1038/mto.2016.24

Figure Lengend Snippet: Optimization of mRNA-EP condition for murine CD8 + T cells and chimeric antigen receptor (CAR) expression profile of mouse mV/m28/m3z CAR-T cells. Murine CD8 + T cells purified from splenocytes and lymph node cells were stimulated with anti-mCD3 and anti-mCD28 mAbs for 96 hours. The activated cells were electroporated with mRNA encoding mV/m28/m3z CAR. ( a ) Summary of mRNA-EP conditions for the optimization study. A CAR-mRNA amount of 100 µg was fixed. ( b ) CAR expression was assessed using immunofluorescent mVEGFR2-Fc chimera and detected by flow cytometric analysis (FCM) at 15 hours after mRNA-EP. The condition of No. 9 was selected as the optimal EP condition for murine CD8 + T cells. ( c ) CAR expression was assessed using immunofluorescent anti-HA antibody and detected by FCM at the indicated time after mRNA-EP under the condition of No. 9. The representative of three experiments is shown. In case of Rv transduction, murine splenocytes and lymph node cells were stimulated with anti-CD3 and anti-CD28 mAbs for 24 hours, and then the activated cells were transduced for 48 hours with Rv containing mV/m28/m3z CAR on a RetroNectin/anti-CD3/CD28 mAbs-coated plate. CAR expression was assessed using immunofluorescent anti-HA antibody and detected by FCM 4 days after Rv transduction. ( b and c ) All cells were pregated Aqua − (live) CD8α + cells. The dotted and solid histograms represent the staining of the mock cells and CAR-T cells, respectively. GMFI ratios were calculated as (GMFI of the CAR-T cells)/(GMFI of the mock cells) and are shown in parentheses at the left upper part of each histogram panel. Cell viabilities were determined by the trypan blue exclusion test and are shown in brackets at the right upper part of each histogram panel.

Article Snippet: Cells were incubated with anti-murine CD16/CD32 mAb (clone 2.4G2, purified from hybridoma supernatants) or human FcR Blocking Reagent (Miltenyi Biotec), and then stained with Live/Dead Fixable Aqua viability dye (Thermo Fisher Scientific), fluorescent-labeled mAbs for anti-murine CD8α (clone 53-6.7, eBioscience, San Diego, CA), anti-hCD8α (clone OKT-8, eBioscience), anti-human CD45RA (clone HI100, eBioscience), anti-human CD45RO (clone UCHL1, BioLegend, San Diego, CA), anti-human CD44 (clone G44-26, eBioscience), anti-human PD-1 (clone eBioJ105, eBioscience), anti-human CD122 mAb (clone TU27, BioLegend), anti-human CD127 (clone eBioRDR5, eBioscience), anti-human CD62L (clone DREG-56, BioLegend), and anti-human CCR7 (clone 150503, BD Biosciences, San Jose, CA) in phosphate buffer saline (PBS) containing 0.5% FBS and 0.05% sodium azide.

Techniques: Expressing, Purification, Transduction, Staining

Journal: Molecular Therapy Oncolytics

Article Title: Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research

doi: 10.1038/mto.2016.24

Figure Lengend Snippet: Optimization of mRNA-EP condition for human CD8 + T cells and chimeric antigen receptor (CAR) expression profile of human CAR-T cells. PBMCs provided by volunteers were cultured with anti-hCD3 mAb-stimulation in media supplemented with recombinant hIL-2 and autologous plasma for 13 days. Human CD8 + T cells purified by MACS were electroporated with mRNA encoding hV/h28/h3z, hV/h8a/h137/h3z, mV/h28/h3z, or mV/h8a/h137/h3z. ( a ) Summary of mRNA-EP conditions for the optimization study. An interval between P P and D P of 50 ms, a D P voltage of 50 V, a D P frequency of three times, a D P duration of 50 ms, and a D P interval of 50 ms were fixed. ( b ) hV/h28/h3z CAR expression was assessed using immunofluorescent anti-HA antibody and detected by flow cytometric analysis (FCM) at 15 hours after mRNA-EP. The condition of No. 1 was selected as the optimal EP condition for human CD8 + T cells. ( c ) CAR expression was assessed using immunofluorescent anti-HA antibody and detected by FCM at the indicated time after mRNA-EP under the condition of No. 1. The representative of three experiments is shown. ( d ) Expression and antigen-binding ability of hV/h28/h3z CAR were assessed using immunofluorescent hVEGFR2-Fc chimera and detected by FCM at 36 hours after mRNA-EP under the condition of No. 1. ( b–d ) All cells were pregated Aqua − (live) CD8α + cells. The dotted and solid histograms represent the staining of the mock cells and CAR-T cells, respectively. Geometric mean fluorescence intensity (GMFI) ratios were calculated as (GMFI of the CAR-T cells)/(GMFI of the mock cells) and are shown in parentheses at the left upper part of each histogram panel. Cell viabilities were determined by the trypan blue exclusion test and are shown in brackets at the right upper part of each histogram panel. NT, not tested.

Article Snippet: Cells were incubated with anti-murine CD16/CD32 mAb (clone 2.4G2, purified from hybridoma supernatants) or human FcR Blocking Reagent (Miltenyi Biotec), and then stained with Live/Dead Fixable Aqua viability dye (Thermo Fisher Scientific), fluorescent-labeled mAbs for anti-murine CD8α (clone 53-6.7, eBioscience, San Diego, CA), anti-hCD8α (clone OKT-8, eBioscience), anti-human CD45RA (clone HI100, eBioscience), anti-human CD45RO (clone UCHL1, BioLegend, San Diego, CA), anti-human CD44 (clone G44-26, eBioscience), anti-human PD-1 (clone eBioJ105, eBioscience), anti-human CD122 mAb (clone TU27, BioLegend), anti-human CD127 (clone eBioRDR5, eBioscience), anti-human CD62L (clone DREG-56, BioLegend), and anti-human CCR7 (clone 150503, BD Biosciences, San Jose, CA) in phosphate buffer saline (PBS) containing 0.5% FBS and 0.05% sodium azide.

Techniques: Expressing, Cell Culture, Recombinant, Purification, Binding Assay, Staining, Fluorescence

Journal: Molecular Therapy Oncolytics

Article Title: Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research

doi: 10.1038/mto.2016.24

Figure Lengend Snippet: Phenotypic analysis at each step of chimeric antigen receptor (CAR)-T cell preparation using mRNA-EP. PBMCs, expanded PBMCs, MACS-purified CD8 + T cells, and CAR-T cells 6 hours after mRNA-EP were stained with fluorescent immunoreagents for each surface molecule. Immunofluorescence, which was analyzed as the relative log fluorescence of gated Aqua − (live) CD8α + cells, was measured by flow cytometric analysis (FCM). ( a ) The percentage shown in each dot plot represents the quadrant frequency. ( b ) The value shown in the right part of each histogram represents the percentage of geometric mean fluorescence intensity to PBMC group. The representative of three experiments is shown.

Article Snippet: Cells were incubated with anti-murine CD16/CD32 mAb (clone 2.4G2, purified from hybridoma supernatants) or human FcR Blocking Reagent (Miltenyi Biotec), and then stained with Live/Dead Fixable Aqua viability dye (Thermo Fisher Scientific), fluorescent-labeled mAbs for anti-murine CD8α (clone 53-6.7, eBioscience, San Diego, CA), anti-hCD8α (clone OKT-8, eBioscience), anti-human CD45RA (clone HI100, eBioscience), anti-human CD45RO (clone UCHL1, BioLegend, San Diego, CA), anti-human CD44 (clone G44-26, eBioscience), anti-human PD-1 (clone eBioJ105, eBioscience), anti-human CD122 mAb (clone TU27, BioLegend), anti-human CD127 (clone eBioRDR5, eBioscience), anti-human CD62L (clone DREG-56, BioLegend), and anti-human CCR7 (clone 150503, BD Biosciences, San Jose, CA) in phosphate buffer saline (PBS) containing 0.5% FBS and 0.05% sodium azide.

Techniques: Purification, Staining, Immunofluorescence, Fluorescence

Journal: Molecular Therapy Oncolytics

Article Title: Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research

doi: 10.1038/mto.2016.24

Figure Lengend Snippet: Antigen-specific phenotypic changes of hVEGFR2-specific chimeric antigen receptor (CAR)-T cells prepared by mRNA-EP. hV/h28/h3z or hV/h8a/h137/h3z CAR-T cells 6 hours after mRNA-EP were stimulated with anti-hCD3 mAb, hVEGFR2-Fc chimera, or mVEGFR2-Fc chimera for 24 hours. Cells were stained with fluorescent immunoreagents for each surface molecule. Immunofluorescence, which was analyzed as the relative log fluorescence of gated Aqua − (live) CD8α + cells, was measured by flow cytometric analysis. ( a ) The percentage shown in each dot plot represents the quadrant frequency. ( b ) The value shown in the right part of each histogram represents the percentage of geometric mean fluorescence intensity to the group of before stimulation. The representative of three experiments is shown.

Article Snippet: Cells were incubated with anti-murine CD16/CD32 mAb (clone 2.4G2, purified from hybridoma supernatants) or human FcR Blocking Reagent (Miltenyi Biotec), and then stained with Live/Dead Fixable Aqua viability dye (Thermo Fisher Scientific), fluorescent-labeled mAbs for anti-murine CD8α (clone 53-6.7, eBioscience, San Diego, CA), anti-hCD8α (clone OKT-8, eBioscience), anti-human CD45RA (clone HI100, eBioscience), anti-human CD45RO (clone UCHL1, BioLegend, San Diego, CA), anti-human CD44 (clone G44-26, eBioscience), anti-human PD-1 (clone eBioJ105, eBioscience), anti-human CD122 mAb (clone TU27, BioLegend), anti-human CD127 (clone eBioRDR5, eBioscience), anti-human CD62L (clone DREG-56, BioLegend), and anti-human CCR7 (clone 150503, BD Biosciences, San Jose, CA) in phosphate buffer saline (PBS) containing 0.5% FBS and 0.05% sodium azide.

Techniques: Staining, Immunofluorescence, Fluorescence

Journal: Molecular Therapy Oncolytics

Article Title: Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research

doi: 10.1038/mto.2016.24

Figure Lengend Snippet: Antigen-specific cytokine production of hVEGFR2-specific chimeric antigen receptor (CAR)-T cells prepared by mRNA-EP. hV/h28/h3z or hV/h8a/h137/h3z CAR-T cells 6 hours after mRNA-EP were stimulated with anti-hCD3 mAb, hVEGFR2-Fc chimera, or mVEGFR2-Fc chimera for the indicated hours ( a ) or 24 hours ( b ). ( a ) Cells were stained with fluorescent immunoreagents for each intracellular cytokine or CD107a. Immunofluorescence, which was analyzed as the relative log fluorescence of gated Aqua − (live) CD8α + cells, was measured by flow cytometric analysis. The pie chart shows the frequency of multifunctional CAR-T cells, and the donut chart shows the frequency of each factor-positive cell. The representative of two experiments is shown. ( b ) The secretion amount of IFN-γ, TNF-α, IL-2, or granzyme-B from hV/h28/h3z (white bar) or hV/h8a/h137/h3z (black bar) CAR-T cells after 24-hour stimulation was determined by ELISA. Data are expressed as the mean ± SD of three cultures. Statistical analysis was performed using one-way analysis of variance: ** P < 0.01.

Article Snippet: Cells were incubated with anti-murine CD16/CD32 mAb (clone 2.4G2, purified from hybridoma supernatants) or human FcR Blocking Reagent (Miltenyi Biotec), and then stained with Live/Dead Fixable Aqua viability dye (Thermo Fisher Scientific), fluorescent-labeled mAbs for anti-murine CD8α (clone 53-6.7, eBioscience, San Diego, CA), anti-hCD8α (clone OKT-8, eBioscience), anti-human CD45RA (clone HI100, eBioscience), anti-human CD45RO (clone UCHL1, BioLegend, San Diego, CA), anti-human CD44 (clone G44-26, eBioscience), anti-human PD-1 (clone eBioJ105, eBioscience), anti-human CD122 mAb (clone TU27, BioLegend), anti-human CD127 (clone eBioRDR5, eBioscience), anti-human CD62L (clone DREG-56, BioLegend), and anti-human CCR7 (clone 150503, BD Biosciences, San Jose, CA) in phosphate buffer saline (PBS) containing 0.5% FBS and 0.05% sodium azide.

Techniques: Staining, Immunofluorescence, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy Oncolytics

Article Title: Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research

doi: 10.1038/mto.2016.24

Figure Lengend Snippet: Optimization of mRNA-EP condition for murine CD8 + T cells and chimeric antigen receptor (CAR) expression profile of mouse mV/m28/m3z CAR-T cells. Murine CD8 + T cells purified from splenocytes and lymph node cells were stimulated with anti-mCD3 and anti-mCD28 mAbs for 96 hours. The activated cells were electroporated with mRNA encoding mV/m28/m3z CAR. ( a ) Summary of mRNA-EP conditions for the optimization study. A CAR-mRNA amount of 100 µg was fixed. ( b ) CAR expression was assessed using immunofluorescent mVEGFR2-Fc chimera and detected by flow cytometric analysis (FCM) at 15 hours after mRNA-EP. The condition of No. 9 was selected as the optimal EP condition for murine CD8 + T cells. ( c ) CAR expression was assessed using immunofluorescent anti-HA antibody and detected by FCM at the indicated time after mRNA-EP under the condition of No. 9. The representative of three experiments is shown. In case of Rv transduction, murine splenocytes and lymph node cells were stimulated with anti-CD3 and anti-CD28 mAbs for 24 hours, and then the activated cells were transduced for 48 hours with Rv containing mV/m28/m3z CAR on a RetroNectin/anti-CD3/CD28 mAbs-coated plate. CAR expression was assessed using immunofluorescent anti-HA antibody and detected by FCM 4 days after Rv transduction. ( b and c ) All cells were pregated Aqua − (live) CD8α + cells. The dotted and solid histograms represent the staining of the mock cells and CAR-T cells, respectively. GMFI ratios were calculated as (GMFI of the CAR-T cells)/(GMFI of the mock cells) and are shown in parentheses at the left upper part of each histogram panel. Cell viabilities were determined by the trypan blue exclusion test and are shown in brackets at the right upper part of each histogram panel.

Article Snippet: Cells were incubated with anti-murine CD16/CD32 mAb (clone 2.4G2, purified from hybridoma supernatants) or human FcR Blocking Reagent (Miltenyi Biotec), and then stained with Live/Dead Fixable Aqua viability dye (Thermo Fisher Scientific), fluorescent-labeled mAbs for anti-murine CD8α (clone 53-6.7, eBioscience, San Diego, CA), anti-hCD8α (clone OKT-8, eBioscience), anti-human CD45RA (clone HI100, eBioscience), anti-human CD45RO (clone UCHL1, BioLegend, San Diego, CA), anti-human CD44 (clone G44-26, eBioscience), anti-human PD-1 (clone eBioJ105, eBioscience), anti-human CD122 mAb (clone TU27, BioLegend), anti-human CD127 (clone eBioRDR5, eBioscience), anti-human CD62L (clone DREG-56, BioLegend), and anti-human CCR7 (clone 150503, BD Biosciences, San Jose, CA) in phosphate buffer saline (PBS) containing 0.5% FBS and 0.05% sodium azide.

Techniques: Expressing, Purification, Transduction, Staining

Journal: Molecular Therapy Oncolytics

Article Title: Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research

doi: 10.1038/mto.2016.24

Figure Lengend Snippet: Optimization of mRNA-EP condition for human CD8 + T cells and chimeric antigen receptor (CAR) expression profile of human CAR-T cells. PBMCs provided by volunteers were cultured with anti-hCD3 mAb-stimulation in media supplemented with recombinant hIL-2 and autologous plasma for 13 days. Human CD8 + T cells purified by MACS were electroporated with mRNA encoding hV/h28/h3z, hV/h8a/h137/h3z, mV/h28/h3z, or mV/h8a/h137/h3z. ( a ) Summary of mRNA-EP conditions for the optimization study. An interval between P P and D P of 50 ms, a D P voltage of 50 V, a D P frequency of three times, a D P duration of 50 ms, and a D P interval of 50 ms were fixed. ( b ) hV/h28/h3z CAR expression was assessed using immunofluorescent anti-HA antibody and detected by flow cytometric analysis (FCM) at 15 hours after mRNA-EP. The condition of No. 1 was selected as the optimal EP condition for human CD8 + T cells. ( c ) CAR expression was assessed using immunofluorescent anti-HA antibody and detected by FCM at the indicated time after mRNA-EP under the condition of No. 1. The representative of three experiments is shown. ( d ) Expression and antigen-binding ability of hV/h28/h3z CAR were assessed using immunofluorescent hVEGFR2-Fc chimera and detected by FCM at 36 hours after mRNA-EP under the condition of No. 1. ( b–d ) All cells were pregated Aqua − (live) CD8α + cells. The dotted and solid histograms represent the staining of the mock cells and CAR-T cells, respectively. Geometric mean fluorescence intensity (GMFI) ratios were calculated as (GMFI of the CAR-T cells)/(GMFI of the mock cells) and are shown in parentheses at the left upper part of each histogram panel. Cell viabilities were determined by the trypan blue exclusion test and are shown in brackets at the right upper part of each histogram panel. NT, not tested.

Article Snippet: Cells were incubated with anti-murine CD16/CD32 mAb (clone 2.4G2, purified from hybridoma supernatants) or human FcR Blocking Reagent (Miltenyi Biotec), and then stained with Live/Dead Fixable Aqua viability dye (Thermo Fisher Scientific), fluorescent-labeled mAbs for anti-murine CD8α (clone 53-6.7, eBioscience, San Diego, CA), anti-hCD8α (clone OKT-8, eBioscience), anti-human CD45RA (clone HI100, eBioscience), anti-human CD45RO (clone UCHL1, BioLegend, San Diego, CA), anti-human CD44 (clone G44-26, eBioscience), anti-human PD-1 (clone eBioJ105, eBioscience), anti-human CD122 mAb (clone TU27, BioLegend), anti-human CD127 (clone eBioRDR5, eBioscience), anti-human CD62L (clone DREG-56, BioLegend), and anti-human CCR7 (clone 150503, BD Biosciences, San Jose, CA) in phosphate buffer saline (PBS) containing 0.5% FBS and 0.05% sodium azide.

Techniques: Expressing, Cell Culture, Recombinant, Purification, Binding Assay, Staining, Fluorescence

Journal: Molecular Therapy Oncolytics

Article Title: Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research

doi: 10.1038/mto.2016.24

Figure Lengend Snippet: Antigen-specific phenotypic changes of hVEGFR2-specific chimeric antigen receptor (CAR)-T cells prepared by mRNA-EP. hV/h28/h3z or hV/h8a/h137/h3z CAR-T cells 6 hours after mRNA-EP were stimulated with anti-hCD3 mAb, hVEGFR2-Fc chimera, or mVEGFR2-Fc chimera for 24 hours. Cells were stained with fluorescent immunoreagents for each surface molecule. Immunofluorescence, which was analyzed as the relative log fluorescence of gated Aqua − (live) CD8α + cells, was measured by flow cytometric analysis. ( a ) The percentage shown in each dot plot represents the quadrant frequency. ( b ) The value shown in the right part of each histogram represents the percentage of geometric mean fluorescence intensity to the group of before stimulation. The representative of three experiments is shown.

Article Snippet: Cells were incubated with anti-murine CD16/CD32 mAb (clone 2.4G2, purified from hybridoma supernatants) or human FcR Blocking Reagent (Miltenyi Biotec), and then stained with Live/Dead Fixable Aqua viability dye (Thermo Fisher Scientific), fluorescent-labeled mAbs for anti-murine CD8α (clone 53-6.7, eBioscience, San Diego, CA), anti-hCD8α (clone OKT-8, eBioscience), anti-human CD45RA (clone HI100, eBioscience), anti-human CD45RO (clone UCHL1, BioLegend, San Diego, CA), anti-human CD44 (clone G44-26, eBioscience), anti-human PD-1 (clone eBioJ105, eBioscience), anti-human CD122 mAb (clone TU27, BioLegend), anti-human CD127 (clone eBioRDR5, eBioscience), anti-human CD62L (clone DREG-56, BioLegend), and anti-human CCR7 (clone 150503, BD Biosciences, San Jose, CA) in phosphate buffer saline (PBS) containing 0.5% FBS and 0.05% sodium azide.

Techniques: Staining, Immunofluorescence, Fluorescence

Journal: Molecular Therapy Oncolytics

Article Title: Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research

doi: 10.1038/mto.2016.24

Figure Lengend Snippet: Antigen-specific cytokine production of hVEGFR2-specific chimeric antigen receptor (CAR)-T cells prepared by mRNA-EP. hV/h28/h3z or hV/h8a/h137/h3z CAR-T cells 6 hours after mRNA-EP were stimulated with anti-hCD3 mAb, hVEGFR2-Fc chimera, or mVEGFR2-Fc chimera for the indicated hours ( a ) or 24 hours ( b ). ( a ) Cells were stained with fluorescent immunoreagents for each intracellular cytokine or CD107a. Immunofluorescence, which was analyzed as the relative log fluorescence of gated Aqua − (live) CD8α + cells, was measured by flow cytometric analysis. The pie chart shows the frequency of multifunctional CAR-T cells, and the donut chart shows the frequency of each factor-positive cell. The representative of two experiments is shown. ( b ) The secretion amount of IFN-γ, TNF-α, IL-2, or granzyme-B from hV/h28/h3z (white bar) or hV/h8a/h137/h3z (black bar) CAR-T cells after 24-hour stimulation was determined by ELISA. Data are expressed as the mean ± SD of three cultures. Statistical analysis was performed using one-way analysis of variance: ** P < 0.01.

Article Snippet: Cells were incubated with anti-murine CD16/CD32 mAb (clone 2.4G2, purified from hybridoma supernatants) or human FcR Blocking Reagent (Miltenyi Biotec), and then stained with Live/Dead Fixable Aqua viability dye (Thermo Fisher Scientific), fluorescent-labeled mAbs for anti-murine CD8α (clone 53-6.7, eBioscience, San Diego, CA), anti-hCD8α (clone OKT-8, eBioscience), anti-human CD45RA (clone HI100, eBioscience), anti-human CD45RO (clone UCHL1, BioLegend, San Diego, CA), anti-human CD44 (clone G44-26, eBioscience), anti-human PD-1 (clone eBioJ105, eBioscience), anti-human CD122 mAb (clone TU27, BioLegend), anti-human CD127 (clone eBioRDR5, eBioscience), anti-human CD62L (clone DREG-56, BioLegend), and anti-human CCR7 (clone 150503, BD Biosciences, San Jose, CA) in phosphate buffer saline (PBS) containing 0.5% FBS and 0.05% sodium azide.

Techniques: Staining, Immunofluorescence, Fluorescence, Enzyme-linked Immunosorbent Assay