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cd11b negative fraction (Miltenyi Biotec)

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Miltenyi Biotec cd11b negative fraction
Cd11b Negative Fraction, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 98/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/cd11b+negative+fraction/CD11b%2Fc+Antibody%2C+anti-rat%2C+REAfinity/pmc12616584-249-9-23
Average 98 stars, based on 26 article reviews

cd11b negative fraction - by Bioz Stars, 2026-09

98/100 stars

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Isolation:

Article Title: Protective exercise responses in the dentate gyrus of Alzheimer’s disease mouse model revealed with single-nucleus sequencing
Article Snippet: CD11b magnetic labeling and separation was performed as in Bordt et al. 2020, using CD11b (Microglia) MicroBeads (#130-093-636, Miltenyi Biotec) LS Columns (130-042-401) and QuadroMACS Separator (130-090-976) after debris/myelin removal using the Debris Removal solution (#130-109-398, Miltenyi Biotec) 102 . .. The CD11b negative fraction was further processed using the mouse Anti-ACSA-2 MicroBead Kit (#130-097-678, Miltenyi Biotec) for the isolation of astrocytes. .. The following lentiviral p.LKO1-puro vectors were procured from Sigma: MISSION ® Non-Target shRNA Control (#SHC016), ATP6V0C-TRCN0000101611; ATPIF1-TRCN0000190288; SLC25A4-TRCN0000069153; NME7-TRCN0000024704; THRA-TRCN0000027109; ST7-TRCN0000179803.

Article Title: Microglial cathepsin E plays a role in neuroinflammation and amyloid β production in Alzheimer’s disease
Article Snippet: Tissue debris were removed by passing the cell suspension through a 30μm cell strainer and magnetically labeled with CD11b microbeads, the cells were extensively washed and separated in a magnetic field using MS columns (Miltenyi Biotec), and the CD11b‐positive fraction was collected. .. CD11b‐negative fraction was collected for further neuron purification using Neuron Isolation Kit, mouse (Miltenyi Biotec). ..

Purification:

Image Search Results

Inhibition of 2-HG production by AG-881 does not alter the tumor-infiltrating myeloid cell population but results in increased recruitment of CD4 + T-cells. (A) Mice bearing dTG-IDH1 R132H tumors were treated daily with 10 mg/kg AG-881 or vehicle. Levels of 2-HG in the tumor-bearing hemisphere after 14 days of treatment were measured by LC-MS/MS. (B–D) A separate cohort of mice were treated as described in (A), and single-cell suspensions of the tumors were either analyzed by FC to determine the frequency of CD11b + (B) cells among tumor-infiltrating leukocytes or used to isolate CD11b + cells using a column separation protocol (C, D). 2-HG levels were significantly reduced in CD11b + cells from AG-811-treated mice (C); however, their ability to suppress CD8 + ((D), left panel) and CD4 + ((D), right panel) T-cell proliferation ex vivo was unchanged. T-cell proliferation as a result of polyclonal CD3/CD28 activation was measured by dilution of CFSE 72 hours post-coculture initiation. (E) Single-cell suspensions generated in (B) were used to examine the frequency for CD8 + and CD4 + T-cells by FC. In 2-HG quantification and T-cell suppression studies n=3 mice per group. For FC analyses, n=4 in control group and n=5 in AG-881 groups. dTG, double transgenic; FC, flow cytometry; HG, hydroxyglutarate.

Journal: Journal for Immunotherapy of Cancer

Article Title: Inhibition of D-2HG leads to upregulation of a proinflammatory gene signature in a novel HLA-A2/HLA-DR1 transgenic mouse model of IDH1R132H-expressing glioma

doi: 10.1136/jitc-2022-004644

Figure Lengend Snippet: Inhibition of 2-HG production by AG-881 does not alter the tumor-infiltrating myeloid cell population but results in increased recruitment of CD4 + T-cells. (A) Mice bearing dTG-IDH1 R132H tumors were treated daily with 10 mg/kg AG-881 or vehicle. Levels of 2-HG in the tumor-bearing hemisphere after 14 days of treatment were measured by LC-MS/MS. (B–D) A separate cohort of mice were treated as described in (A), and single-cell suspensions of the tumors were either analyzed by FC to determine the frequency of CD11b + (B) cells among tumor-infiltrating leukocytes or used to isolate CD11b + cells using a column separation protocol (C, D). 2-HG levels were significantly reduced in CD11b + cells from AG-811-treated mice (C); however, their ability to suppress CD8 + ((D), left panel) and CD4 + ((D), right panel) T-cell proliferation ex vivo was unchanged. T-cell proliferation as a result of polyclonal CD3/CD28 activation was measured by dilution of CFSE 72 hours post-coculture initiation. (E) Single-cell suspensions generated in (B) were used to examine the frequency for CD8 + and CD4 + T-cells by FC. In 2-HG quantification and T-cell suppression studies n=3 mice per group. For FC analyses, n=4 in control group and n=5 in AG-881 groups. dTG, double transgenic; FC, flow cytometry; HG, hydroxyglutarate.

Article Snippet: The latter was used to separate CD11b + and CD11b neg fractions via the CD11b microbeads magnetic cell sorting kit (Miltenyi Biotec). mRNA was isolated from both whole tumor samples and isolated cell pellets using the RNeasy Plus Mini Kit (Qiagen, #74134).

Techniques: Inhibition, Liquid Chromatography with Mass Spectroscopy, Ex Vivo, Activation Assay, Generated, Control, Transgenic Assay, Flow Cytometry

Prophylactic IDH1 R132H peptide vaccine extends survival only when combined with class I peptide vaccine and IDH1 R132H inhibition. (A) 9-week-old dTG mice received s.c. vaccinations with 100 µg of R132Hp emulsified in IFA and 20 µg intramuscular (i.m.) poly-ICLC on days 0 and 11. On day 21, mice were euthanized, and their spleens were harvested. IFNγ secretion by splenocytes stimulated ex vivo for 72 hours with peptide-pulsed syngeneic CD11b + cells as indicated. Cells from three individual mice were plated in technical triplicates. Media only, no CD11b + cells negative control. (B) Schematic representation of the treatment protocol for prophylactic vaccinations. 9–10 week-old dTG mice received s.c. vaccinations as described in (A) followed by an intracerebral challenge with dTG-IDH1 R132H cells. After 7 days, tumor-bearing mice were randomized to receive daily oral administrations of 10 mg/kg AG-881 or vehicle control for up to 35 consecutive days. Kaplan-Meier curves demonstrate survival outcomes: Sham, Vehicle (n=4); Sham, AG-881 (n=6); R132Hp, Vehicle (n=6), R132Hp, AG-881 (n=6). (C) Diagram representing the retroviral vector used to transduce the dTG-IDH1 R132H cells with HLA class I TAA epitopes derived from gp100, tyrosinase-related protein 2 (Trp2), and tyrosinase (Tyr). (D) Schematic representation of the treatment protocol. 7–10-week-old dTG mice received s.c. vaccinations with the class II R132Hp, peptides gp100 209-217 , Trp2 180-188 , Tyr 368-376 (class I vacc), or PBS (Sham Vacc) emulsified in IFA and 20 µg i.m. poly-ICLC. Vaccines were boosted 10 and 15 days later. And 21 days after initial vaccine administration, all mice received an intracerebral challenge with the dTG-IDH1 R132H ; Class I TAA + cells tumor cells (described in (C)). Seven days later, tumor-bearing mice started receiving daily oral administration of 10 mg/kg AG-881 or vehicle control for up to 35 days. Kaplan-Meier curves demonstrate survival outcomes: Sham, Vehicle (n=4); Class I Vacc, Vehicle (n=4); R132Hp, AG-−881 (n=5), Class I Vacc, AG881 (n=5), and Class I+R132 Hp, AG-881 (n=5). *P<0.05; long-rank test. dTG, double transgenic; HLA, human leukocyte antigens; IFA, incomplete Freund adjuvant; TAA, tumor-associated antigen.

Journal: Journal for Immunotherapy of Cancer

Article Title: Inhibition of D-2HG leads to upregulation of a proinflammatory gene signature in a novel HLA-A2/HLA-DR1 transgenic mouse model of IDH1R132H-expressing glioma

doi: 10.1136/jitc-2022-004644

Figure Lengend Snippet: Prophylactic IDH1 R132H peptide vaccine extends survival only when combined with class I peptide vaccine and IDH1 R132H inhibition. (A) 9-week-old dTG mice received s.c. vaccinations with 100 µg of R132Hp emulsified in IFA and 20 µg intramuscular (i.m.) poly-ICLC on days 0 and 11. On day 21, mice were euthanized, and their spleens were harvested. IFNγ secretion by splenocytes stimulated ex vivo for 72 hours with peptide-pulsed syngeneic CD11b + cells as indicated. Cells from three individual mice were plated in technical triplicates. Media only, no CD11b + cells negative control. (B) Schematic representation of the treatment protocol for prophylactic vaccinations. 9–10 week-old dTG mice received s.c. vaccinations as described in (A) followed by an intracerebral challenge with dTG-IDH1 R132H cells. After 7 days, tumor-bearing mice were randomized to receive daily oral administrations of 10 mg/kg AG-881 or vehicle control for up to 35 consecutive days. Kaplan-Meier curves demonstrate survival outcomes: Sham, Vehicle (n=4); Sham, AG-881 (n=6); R132Hp, Vehicle (n=6), R132Hp, AG-881 (n=6). (C) Diagram representing the retroviral vector used to transduce the dTG-IDH1 R132H cells with HLA class I TAA epitopes derived from gp100, tyrosinase-related protein 2 (Trp2), and tyrosinase (Tyr). (D) Schematic representation of the treatment protocol. 7–10-week-old dTG mice received s.c. vaccinations with the class II R132Hp, peptides gp100 209-217 , Trp2 180-188 , Tyr 368-376 (class I vacc), or PBS (Sham Vacc) emulsified in IFA and 20 µg i.m. poly-ICLC. Vaccines were boosted 10 and 15 days later. And 21 days after initial vaccine administration, all mice received an intracerebral challenge with the dTG-IDH1 R132H ; Class I TAA + cells tumor cells (described in (C)). Seven days later, tumor-bearing mice started receiving daily oral administration of 10 mg/kg AG-881 or vehicle control for up to 35 days. Kaplan-Meier curves demonstrate survival outcomes: Sham, Vehicle (n=4); Class I Vacc, Vehicle (n=4); R132Hp, AG-−881 (n=5), Class I Vacc, AG881 (n=5), and Class I+R132 Hp, AG-881 (n=5). *P<0.05; long-rank test. dTG, double transgenic; HLA, human leukocyte antigens; IFA, incomplete Freund adjuvant; TAA, tumor-associated antigen.

Techniques: Inhibition, Ex Vivo, Negative Control, Control, Retroviral, Plasmid Preparation, Transduction, Derivative Assay, Vaccines, Transgenic Assay, Adjuvant

( A ) Flow cytometric analysis demonstrating CD11b + Gr-1 + MDSC accumulation (% CD45 + leukocytes, top ) and L-selectin expression on CD4 + CD44 lo and CD8 + CD44 lo naïve T cells ( below ) in the indicated lymphoid organs (thymus, peripheral lymph node (pLN), inguinal (Ing) LN, tumor-draining Ing LN, spleen, and blood) of non-tumor bearing (NTB) mice or 4T1-bearing mice (tumor volume 1150 ± 150 mm 3 ). ( B ) MDSC-T cell suppression assay. Splenic CD11b + cells either from NTB mice or 4T1-bearing mice (tumor volume 2600 ± 380 mm 3 ) were co-cultured with CFSE-labeled target splenocytes from NTB mice at the indicated splenocyte:myeloid cell ratios. Proliferation (based on CFSE dilution) in T cell subsets was measured 72 hr after addition of anti-CD3/CD28 antibody-conjugated activation beads. Percent suppression is for one experiment (mean±s.e.m, n = 3 replicates per condition) and is representative of three independent experiments. ( C ) Total numbers of viable naïve CD4 + CD44 lo and CD8 + CD44 lo T cell subsets ( left ) and percentages of annexin V + early apoptotic T cells ( right ) were quantified by flow cytometric analysis from peripheral lymph nodes and spleens of NTB or 4T1-bearing mice (tumor volume 1340 ± 242 mm 3 ). Data (mean±s.e.m.) are of one experiment ( n = 3 mice per group) and are representative of two independent experiments. ( B , C ) *p<0.05; ns, not significant; data were analyzed by unpaired two-tailed Student’s t -test. ( D ) Splenic cryosections from NTB and 4T1-bearing mice stained for B220 + , Gr-1 + and CD3 + cells; parallel fluorocytometric analysis (as in A ) confirmed that >90% of splenic Gr-1 + cells co-expressed CD11b. Scale bar, 50 µm. ( E ) L-selectin expression on splenic B220 + cells of NTB and 4T1–bearing mice. ( A , E ) Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. ( A , B , D–E ) Data are for one experiment and are representative of ≥ three independent experiments ( n = 3 replicates or mice per group). pLN, peripheral lymph node; Ing LN, inguinal lymph node; NTB, non-tumor bearing. DOI: http://dx.doi.org/10.7554/eLife.17375.002

Journal: eLife

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes

doi: 10.7554/eLife.17375

Figure Lengend Snippet: ( A ) Flow cytometric analysis demonstrating CD11b + Gr-1 + MDSC accumulation (% CD45 + leukocytes, top ) and L-selectin expression on CD4 + CD44 lo and CD8 + CD44 lo naïve T cells ( below ) in the indicated lymphoid organs (thymus, peripheral lymph node (pLN), inguinal (Ing) LN, tumor-draining Ing LN, spleen, and blood) of non-tumor bearing (NTB) mice or 4T1-bearing mice (tumor volume 1150 ± 150 mm 3 ). ( B ) MDSC-T cell suppression assay. Splenic CD11b + cells either from NTB mice or 4T1-bearing mice (tumor volume 2600 ± 380 mm 3 ) were co-cultured with CFSE-labeled target splenocytes from NTB mice at the indicated splenocyte:myeloid cell ratios. Proliferation (based on CFSE dilution) in T cell subsets was measured 72 hr after addition of anti-CD3/CD28 antibody-conjugated activation beads. Percent suppression is for one experiment (mean±s.e.m, n = 3 replicates per condition) and is representative of three independent experiments. ( C ) Total numbers of viable naïve CD4 + CD44 lo and CD8 + CD44 lo T cell subsets ( left ) and percentages of annexin V + early apoptotic T cells ( right ) were quantified by flow cytometric analysis from peripheral lymph nodes and spleens of NTB or 4T1-bearing mice (tumor volume 1340 ± 242 mm 3 ). Data (mean±s.e.m.) are of one experiment ( n = 3 mice per group) and are representative of two independent experiments. ( B , C ) *p<0.05; ns, not significant; data were analyzed by unpaired two-tailed Student’s t -test. ( D ) Splenic cryosections from NTB and 4T1-bearing mice stained for B220 + , Gr-1 + and CD3 + cells; parallel fluorocytometric analysis (as in A ) confirmed that >90% of splenic Gr-1 + cells co-expressed CD11b. Scale bar, 50 µm. ( E ) L-selectin expression on splenic B220 + cells of NTB and 4T1–bearing mice. ( A , E ) Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. ( A , B , D–E ) Data are for one experiment and are representative of ≥ three independent experiments ( n = 3 replicates or mice per group). pLN, peripheral lymph node; Ing LN, inguinal lymph node; NTB, non-tumor bearing. DOI: http://dx.doi.org/10.7554/eLife.17375.002

Article Snippet: To obtain an enriched CD8 + T cell population (routinely > 90%), the CD11b neg fraction of splenocytes was then subjected to negative selection using a CD8 + magnetic bead separation kit (Miltenyi Biotec; ).

Techniques: Expressing, Suppression Assay, Cell Culture, Labeling, Activation Assay, Two Tailed Test, Staining, Fluorescence

Flow cytometric analysis of L-selectin expression on splenic CD3 + CD44 lo naïve T cells from non-tumor bearing (NTB) mice and CD11b + Gr-1 + MDSC from 4T1-bearing mice (tumor volume 1050 ± 150 mm 3 ). Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity (MFI). Data (mean±s.e.m.) are of one experiment ( n = 3 mice per group) and are representative of two independent experiments. *p<0.05; data were analyzed by unpaired two-tailed Student’s t -test. NTB, non-tumor bearing; MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.17375.003

Journal: eLife

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes

doi: 10.7554/eLife.17375

Figure Lengend Snippet: Flow cytometric analysis of L-selectin expression on splenic CD3 + CD44 lo naïve T cells from non-tumor bearing (NTB) mice and CD11b + Gr-1 + MDSC from 4T1-bearing mice (tumor volume 1050 ± 150 mm 3 ). Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity (MFI). Data (mean±s.e.m.) are of one experiment ( n = 3 mice per group) and are representative of two independent experiments. *p<0.05; data were analyzed by unpaired two-tailed Student’s t -test. NTB, non-tumor bearing; MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.17375.003

Techniques: Expressing, Staining, Fluorescence, Two Tailed Test

( A ) Fluorocytometric analysis of CD11b + Gr-1 + MDSC accumulation (% CD45 + leukocytes, top ) and L-selectin expression on splenic CD4 + CD44 lo and CD8 + CD44 lo naïve T cells ( below ) of non-tumor bearing (NTB) or 4T1-bearing male mice (tumor volume ~2000 mm 3 ). ( B ) Tumor volume ( top left ) and accumulation of splenic CD11b + Gr-1 + MDSC ( bottom left ) in 4T1-tumor-bearing female mice were measured over time. Representative flow cytometric analysis is shown for CD11b + Gr-1 + cell frequency (% CD45 + leukocytes) of NTB controls compared to 28 days post-4T1 implantation ( right ). ( C ) L-selectin on splenic CD4 + CD44 lo and CD8 + CD44 lo naïve T cells in 4T1-bearing female mice was assessed during tumor progression and compared with NTB mice ( left ). Representative L-selectin profiles are shown for NTB and 4T1-bearing mice at 28 days post-tumor implantation ( right ). ( B , C ) Data (mean±s.e.m.) are for one representative experiment ( n = 3 mice per group); *p<0.05; data were analyzed by unpaired two-tailed Student’s t -test. ( D ) Flow cytometric analysis of CD11b + Gr-1 + MDSC (% CD45 + leukocytes, left ) and L-selectin expression profiles on CD4 + and CD8 + T cells ( right ) in spleens of female mice implanted with orthotopic 4T1 tumors in the mammary fat pad (MFP; tumor volume >1500 mm 3 ). ( A , C–D ) Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity (MFI). ( A–D ) Data are of one experiment ( n = 3 mice per group) and are representative of > three independent experiments. NTB, non-tumor bearing; MFP, mammary fat pad; MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.17375.004

Journal: eLife

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes

doi: 10.7554/eLife.17375

Figure Lengend Snippet: ( A ) Fluorocytometric analysis of CD11b + Gr-1 + MDSC accumulation (% CD45 + leukocytes, top ) and L-selectin expression on splenic CD4 + CD44 lo and CD8 + CD44 lo naïve T cells ( below ) of non-tumor bearing (NTB) or 4T1-bearing male mice (tumor volume ~2000 mm 3 ). ( B ) Tumor volume ( top left ) and accumulation of splenic CD11b + Gr-1 + MDSC ( bottom left ) in 4T1-tumor-bearing female mice were measured over time. Representative flow cytometric analysis is shown for CD11b + Gr-1 + cell frequency (% CD45 + leukocytes) of NTB controls compared to 28 days post-4T1 implantation ( right ). ( C ) L-selectin on splenic CD4 + CD44 lo and CD8 + CD44 lo naïve T cells in 4T1-bearing female mice was assessed during tumor progression and compared with NTB mice ( left ). Representative L-selectin profiles are shown for NTB and 4T1-bearing mice at 28 days post-tumor implantation ( right ). ( B , C ) Data (mean±s.e.m.) are for one representative experiment ( n = 3 mice per group); *p<0.05; data were analyzed by unpaired two-tailed Student’s t -test. ( D ) Flow cytometric analysis of CD11b + Gr-1 + MDSC (% CD45 + leukocytes, left ) and L-selectin expression profiles on CD4 + and CD8 + T cells ( right ) in spleens of female mice implanted with orthotopic 4T1 tumors in the mammary fat pad (MFP; tumor volume >1500 mm 3 ). ( A , C–D ) Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity (MFI). ( A–D ) Data are of one experiment ( n = 3 mice per group) and are representative of > three independent experiments. NTB, non-tumor bearing; MFP, mammary fat pad; MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.17375.004

Techniques: Expressing, Tumor Implantation, Two Tailed Test, Staining, Fluorescence

( A ) Tumor volume ( top left ) and accumulation of splenic CD11b + Gr-1 + MDSC ( bottom left ) in AT-3-tumor-bearing mice were measured over time. Representative flow cytometric analysis is shown ( right ) for CD11b + Gr-1 + cell frequency (% CD45 + leukocytes) in the spleens of non-tumor bearing (NTB) controls compared with AT-3 tumors-bearing mice (28 days post-tumor implantation). ( B ) L-selectin on splenic CD4 + CD44 lo and CD8 + CD44 lo naïve T cells in AT3-bearing mice was compared to L-selectin levels on T cells from NTB mice ( left ). Representative L-selectin profiles of NTB and AT-3-bearing mice are shown ( right ). Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity (MFI). ( A , B ) Data (mean±s.e.m.) are from one experiment ( n = 3 mice per group) and are representative of > three independent experiments. *p<0.05; ns, not significant; data were analyzed by unpaired two-tailed Student’s t -test. NTB, non-tumor bearing; MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.17375.005

Journal: eLife

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes

doi: 10.7554/eLife.17375

Figure Lengend Snippet: ( A ) Tumor volume ( top left ) and accumulation of splenic CD11b + Gr-1 + MDSC ( bottom left ) in AT-3-tumor-bearing mice were measured over time. Representative flow cytometric analysis is shown ( right ) for CD11b + Gr-1 + cell frequency (% CD45 + leukocytes) in the spleens of non-tumor bearing (NTB) controls compared with AT-3 tumors-bearing mice (28 days post-tumor implantation). ( B ) L-selectin on splenic CD4 + CD44 lo and CD8 + CD44 lo naïve T cells in AT3-bearing mice was compared to L-selectin levels on T cells from NTB mice ( left ). Representative L-selectin profiles of NTB and AT-3-bearing mice are shown ( right ). Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity (MFI). ( A , B ) Data (mean±s.e.m.) are from one experiment ( n = 3 mice per group) and are representative of > three independent experiments. *p<0.05; ns, not significant; data were analyzed by unpaired two-tailed Student’s t -test. NTB, non-tumor bearing; MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.17375.005

Techniques: Tumor Implantation, Staining, Fluorescence, Two Tailed Test

Flow cytometric analysis of CD11b + Gr-1 + MDSC (% CD45 + leukocytes, top ) and L-selectin expression profiles on CD4 + and CD8 + T cells ( bottom ) in spleens of mice implanted with subcutaneous B16 melanoma and CT26 colorectal tumors (in C57BL/6 mice or BALB/c mice, respectively). Tumor volume >1500 mm 3 for all tumor models. CD11b + Gr-1 + cells in non-tumor bearing (NTB) controls were <3% of CD45 + leukocytes ( data not shown ). Comparative analysis of L-selectin expression is shown for tumor-bearing mice and NTB controls; horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. Data are representative of ≥ three independent experiments. NTB, non-tumor bearing. DOI: http://dx.doi.org/10.7554/eLife.17375.006

Journal: eLife

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes

doi: 10.7554/eLife.17375

Figure Lengend Snippet: Flow cytometric analysis of CD11b + Gr-1 + MDSC (% CD45 + leukocytes, top ) and L-selectin expression profiles on CD4 + and CD8 + T cells ( bottom ) in spleens of mice implanted with subcutaneous B16 melanoma and CT26 colorectal tumors (in C57BL/6 mice or BALB/c mice, respectively). Tumor volume >1500 mm 3 for all tumor models. CD11b + Gr-1 + cells in non-tumor bearing (NTB) controls were <3% of CD45 + leukocytes ( data not shown ). Comparative analysis of L-selectin expression is shown for tumor-bearing mice and NTB controls; horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. Data are representative of ≥ three independent experiments. NTB, non-tumor bearing. DOI: http://dx.doi.org/10.7554/eLife.17375.006

Techniques: Expressing, Staining, Fluorescence

( A ) Experimental design in which splenectomy or sham surgery was performed in non-tumor bearing (NTB) mice. Mice were then inoculated with 4T1 tumor or maintained as NTB controls. Fluorescently-labeled L-selectin hi CD8 + T cells isolated from NTB mice (input) were used for intravenous adoptive cell transfer (ACT) into tumor-bearing mice or in NTB controls. ( B ) 4T1 tumor volume of sham and splenectomized mice at 21 days post-4T1 implantation. Data (mean±s.e.m.) are for a single representative experiment ( n = 3 mice per group); ns, not significant; data were analyzed by unpaired two-tailed Student’s t -test. ( C ) Representative flow cytometric analysis showing accumulation of CD11b + Gr-1 + cells (% CD45 + leukocytes, top ) and L-selectin expression ( bottom ) on CD8 + CD44 lo T cells before ACT (input) and 24 hr post-ACT in the blood of sham or splenectomized NTB and 4T1-bearing recipient mice. Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. ( A–C ) Data are representative of three independent experiments ( n = 3 mice per group). NTB, non-tumor bearing; ACT, adoptive cell transfer. DOI: http://dx.doi.org/10.7554/eLife.17375.007

Journal: eLife

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes

doi: 10.7554/eLife.17375

Figure Lengend Snippet: ( A ) Experimental design in which splenectomy or sham surgery was performed in non-tumor bearing (NTB) mice. Mice were then inoculated with 4T1 tumor or maintained as NTB controls. Fluorescently-labeled L-selectin hi CD8 + T cells isolated from NTB mice (input) were used for intravenous adoptive cell transfer (ACT) into tumor-bearing mice or in NTB controls. ( B ) 4T1 tumor volume of sham and splenectomized mice at 21 days post-4T1 implantation. Data (mean±s.e.m.) are for a single representative experiment ( n = 3 mice per group); ns, not significant; data were analyzed by unpaired two-tailed Student’s t -test. ( C ) Representative flow cytometric analysis showing accumulation of CD11b + Gr-1 + cells (% CD45 + leukocytes, top ) and L-selectin expression ( bottom ) on CD8 + CD44 lo T cells before ACT (input) and 24 hr post-ACT in the blood of sham or splenectomized NTB and 4T1-bearing recipient mice. Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. ( A–C ) Data are representative of three independent experiments ( n = 3 mice per group). NTB, non-tumor bearing; ACT, adoptive cell transfer. DOI: http://dx.doi.org/10.7554/eLife.17375.007

Techniques: Labeling, Isolation, Two Tailed Test, Expressing, Staining, Fluorescence

Splenectomy or sham surgery was performed 10 days prior to 4T1 tumor inoculation and tissues were evaluated for MDSC expansion and L-selectin expression 22 days after tumor implantation (tumor volume ~1000 mm 3 ). Flow cytometric analysis of CD11b + Gr-1 + cell burden (% CD45 + leukocytes; top ) and L-selectin expression on endogenous B220 + B cell populations ( bottom ) in the indicated organs (BM, bone marrow; spleen; blood) of sham or splenectomized non-tumor bearing (NTB) and 4T1-tumor bearing mice. Horizontal lines on histograms indicate positively stained cells; numbers are mean fluorescence intensity. Data are representative of ≥ three independent experiments ( n = 3 mice per group); NTB, non-tumor bearing; BM, bone marrow. DOI: http://dx.doi.org/10.7554/eLife.17375.009

Journal: eLife

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes

doi: 10.7554/eLife.17375

Figure Lengend Snippet: Splenectomy or sham surgery was performed 10 days prior to 4T1 tumor inoculation and tissues were evaluated for MDSC expansion and L-selectin expression 22 days after tumor implantation (tumor volume ~1000 mm 3 ). Flow cytometric analysis of CD11b + Gr-1 + cell burden (% CD45 + leukocytes; top ) and L-selectin expression on endogenous B220 + B cell populations ( bottom ) in the indicated organs (BM, bone marrow; spleen; blood) of sham or splenectomized non-tumor bearing (NTB) and 4T1-tumor bearing mice. Horizontal lines on histograms indicate positively stained cells; numbers are mean fluorescence intensity. Data are representative of ≥ three independent experiments ( n = 3 mice per group); NTB, non-tumor bearing; BM, bone marrow. DOI: http://dx.doi.org/10.7554/eLife.17375.009

Techniques: Expressing, Tumor Implantation, Staining, Fluorescence

Schematic is shown for experimental design in which 4T1-bearing mice underwent splenectomy or sham surgery 14 days-post tumor implantation. Fluorescently-labeled CD8 + T cells isolated from NTB mice (input) were intravenously transferred 21 days post-4T1 inoculation (tumor volume 1499 ± 449 mm 3 , n = 3 mice). Representative flow cytometric dot plots demonstrating CD11b + Gr-1 + MDSC accumulation in blood (% of CD45 + leukocytes, top ) and histograms ( below ) for L-selectin expression on CD8 + CD44 lo T cells before adoptive cell transfer (ACT) (input) and on circulating transferred T cells 24 hr post-ACT in sham and splenectomized NTB recipients or 4T1-bearing recipients. L-selectin profiles are also shown for endogenous B220 + B cells in spleen and blood of sham and splenectomized NTB mice and 4T1-tumor bearing mice ( bottom histograms). Data are from one experiment ( n = 3 mice per group). Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. NTB, non-tumor bearing; ACT, adoptive cell transfer. DOI: http://dx.doi.org/10.7554/eLife.17375.010

Journal: eLife

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes

doi: 10.7554/eLife.17375

Figure Lengend Snippet: Schematic is shown for experimental design in which 4T1-bearing mice underwent splenectomy or sham surgery 14 days-post tumor implantation. Fluorescently-labeled CD8 + T cells isolated from NTB mice (input) were intravenously transferred 21 days post-4T1 inoculation (tumor volume 1499 ± 449 mm 3 , n = 3 mice). Representative flow cytometric dot plots demonstrating CD11b + Gr-1 + MDSC accumulation in blood (% of CD45 + leukocytes, top ) and histograms ( below ) for L-selectin expression on CD8 + CD44 lo T cells before adoptive cell transfer (ACT) (input) and on circulating transferred T cells 24 hr post-ACT in sham and splenectomized NTB recipients or 4T1-bearing recipients. L-selectin profiles are also shown for endogenous B220 + B cells in spleen and blood of sham and splenectomized NTB mice and 4T1-tumor bearing mice ( bottom histograms). Data are from one experiment ( n = 3 mice per group). Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. NTB, non-tumor bearing; ACT, adoptive cell transfer. DOI: http://dx.doi.org/10.7554/eLife.17375.010

Techniques: Tumor Implantation, Labeling, Isolation, Expressing, Staining, Fluorescence

Schematic is shown for experiment design in which fluorescently-labeled, L-selectin hi splenocytes isolated from non-tumor bearing donors (input) were adoptively transferred into 4T1-bearing recipient mice (tumor volume 1026 ± 54 mm 3 , n = 2 mice). Representative flow cytometric dot plots demonstrating CD11b + Gr-1 + MDSC accumulation in blood (% CD45 + leukocytes, left ) and histograms for L-selectin expression ( right ) on CD8 + CD44 lo , CD4 + CD44 lo , and B220 + splenocytes before adoptive cell transfer (ACT) (input) and on circulating transferred lymphocyte populations 2 hr post-ACT into 4T1-bearing recipient mice. Data are from one experiment ( n = 2 mice per group). Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. NTB, non-tumor bearing; ACT, adoptive cell transfer. DOI: http://dx.doi.org/10.7554/eLife.17375.011

Journal: eLife

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes

doi: 10.7554/eLife.17375

Figure Lengend Snippet: Schematic is shown for experiment design in which fluorescently-labeled, L-selectin hi splenocytes isolated from non-tumor bearing donors (input) were adoptively transferred into 4T1-bearing recipient mice (tumor volume 1026 ± 54 mm 3 , n = 2 mice). Representative flow cytometric dot plots demonstrating CD11b + Gr-1 + MDSC accumulation in blood (% CD45 + leukocytes, left ) and histograms for L-selectin expression ( right ) on CD8 + CD44 lo , CD4 + CD44 lo , and B220 + splenocytes before adoptive cell transfer (ACT) (input) and on circulating transferred lymphocyte populations 2 hr post-ACT into 4T1-bearing recipient mice. Data are from one experiment ( n = 2 mice per group). Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. NTB, non-tumor bearing; ACT, adoptive cell transfer. DOI: http://dx.doi.org/10.7554/eLife.17375.011

Techniques: Labeling, Isolation, Expressing, Staining, Fluorescence

Blood was collected from individual transgenic MTAG mice (with the indicated total tumor volume) and from age-matched non-tumor bearing (NTB) wildtype littermates. Flow cytometric analysis is shown for CD11b + Gr-1 + cell accumulation (% CD45 + leukocyte, above ) and L-selectin expression on naive CD4 + CD44 lo and CD8 + CD44 lo T cells and B220 + B cells ( below ). Data are from one experiment ( n ≥ 3 mice per group) and are representative of three independent experiments. Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. NTB, non-tumor bearing. DOI: http://dx.doi.org/10.7554/eLife.17375.012

Journal: eLife

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes

doi: 10.7554/eLife.17375

Figure Lengend Snippet: Blood was collected from individual transgenic MTAG mice (with the indicated total tumor volume) and from age-matched non-tumor bearing (NTB) wildtype littermates. Flow cytometric analysis is shown for CD11b + Gr-1 + cell accumulation (% CD45 + leukocyte, above ) and L-selectin expression on naive CD4 + CD44 lo and CD8 + CD44 lo T cells and B220 + B cells ( below ). Data are from one experiment ( n ≥ 3 mice per group) and are representative of three independent experiments. Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. NTB, non-tumor bearing. DOI: http://dx.doi.org/10.7554/eLife.17375.012

Techniques: Transgenic Assay, Expressing, Staining, Fluorescence

Normal human-donor peripheral blood lymphocytes were adoptively transferred into non-tumor bearing (NTB) severe-combined immunodeficient (SCID) mice or 4T1-bearing SCID mice at 21 days-post tumor implantation (average tumor volume for three experiments, 1190 ± 197 mm 3 ). The burden of CD11b + Gr-1 + cells in the blood of NTB and 4T1-bearing SCID mice was measured 24 hr after adoptive cell transfer ( top ) and was quantified based on blood volume for T- and B-cell deficient SCID mice. Data (mean±s.e.m.) are from three independent experiments for three different lymphocyte donors ( n = 3 mice per group in each experiment). *p<0.05; data were analyzed by unpaired two-tailed Student’s t -test. Flow histograms depict L-selectin expression of individual donor CD3 + CD45RA + T cells at 24 hr post-ACT in NTB or 4T1 tumor-bearing recipient SCID mice ( bottom ). Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. NTB, non-tumor bearing. DOI: http://dx.doi.org/10.7554/eLife.17375.013

Journal: eLife

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes

doi: 10.7554/eLife.17375

Figure Lengend Snippet: Normal human-donor peripheral blood lymphocytes were adoptively transferred into non-tumor bearing (NTB) severe-combined immunodeficient (SCID) mice or 4T1-bearing SCID mice at 21 days-post tumor implantation (average tumor volume for three experiments, 1190 ± 197 mm 3 ). The burden of CD11b + Gr-1 + cells in the blood of NTB and 4T1-bearing SCID mice was measured 24 hr after adoptive cell transfer ( top ) and was quantified based on blood volume for T- and B-cell deficient SCID mice. Data (mean±s.e.m.) are from three independent experiments for three different lymphocyte donors ( n = 3 mice per group in each experiment). *p<0.05; data were analyzed by unpaired two-tailed Student’s t -test. Flow histograms depict L-selectin expression of individual donor CD3 + CD45RA + T cells at 24 hr post-ACT in NTB or 4T1 tumor-bearing recipient SCID mice ( bottom ). Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. NTB, non-tumor bearing. DOI: http://dx.doi.org/10.7554/eLife.17375.013

Techniques: Tumor Implantation, Two Tailed Test, Expressing, Staining, Fluorescence

( A ) 4T1-tumor-bearing mice were treated with anti-Gr-1 antibodies (α-Gr-1 Ab) or isotype control antibodies (Iso Ab) every 3 days for three weeks starting at three days post-tumor implantation. Endpoint tumor volumes are shown. ( B ) CD11b + Gr-1 + MDSC burden (% CD45 + leukocytes, left ) and L-selectin expression (mean fluorescence intensity, MFI) on endogenous CD4 + CD44 lo , CD8 + CD44 lo , and B220 + lymphocytes ( right ) were measured in the blood of NTB or in 4T1-bearing mice treated with Iso Ab or anti-Gr-1 Ab. ( C ) Splenocytes from NTB or 4T1-bearing mice were depleted of CD11b + cells by magnetic bead isolation (94.8 ± 1.8% depletion, n = 3 mice). These cell populations were then fluorescently-labeled with different tracking dyes, co-mixed at a 1:1 ratio, and cultured in vitro or adoptively transferred into NTB recipients. Representative flow cytometric L-selectin profiles are shown for naïve CD4 + CD44 lo and CD8 + CD44 lo T cells before culture or adoptive cell transfer (ACT) (input) and four days after in culture (in vitro) or for cells recovered from blood and spleen post-ACT ( left ). Quantification of L-selectin modulation ( right ) is based on a ratio of the MFI for T cells from 4T1-bearing mice relative to NTB mice; dashed lines indicate NTB control. ( D ) MDSC or CD11b + control cells were isolated from 4T1-tumor bearing mice (tumor volume >1000 mm 3 ) or NTB mice, respectively. Myeloid cells were then co-cultured with fluorescently-labeled splenocytes from NTB mice (10:1 ratio) in media alone or with IFN-γ (20 U/mL) and LPS (100 ng/mL). MDSC and splenocytes were separated by transwell inserts (0.4 μm pore size) in the indicated co-cultures. After 24 hr, L-selectin expression on viable naive CD8 + CD44 lo T cells was analyzed by flow cytometry; representative profiles are shown ( left ). Relative changes in L-selectin expression were normalized to untreated CD8 + CD44 lo T cells (indicated by dashed lines; right ). ( A–D ) Data (mean±s.e.m.) are from one experiment ( n = 3 mice per group or ≥3 replicates per group) and are representative of ≥ two independent experiments. *p<0.05; ns, not significant; data were analyzed by unpaired two-tailed Student’s t -test. ( C , D ) Horizontal lines in histograms indicate positively stained cells; numbers are MFI. NTB, non-tumor bearing; Ab, antibody; Iso, isotype; MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.17375.014

Journal: eLife

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes

doi: 10.7554/eLife.17375

Figure Lengend Snippet: ( A ) 4T1-tumor-bearing mice were treated with anti-Gr-1 antibodies (α-Gr-1 Ab) or isotype control antibodies (Iso Ab) every 3 days for three weeks starting at three days post-tumor implantation. Endpoint tumor volumes are shown. ( B ) CD11b + Gr-1 + MDSC burden (% CD45 + leukocytes, left ) and L-selectin expression (mean fluorescence intensity, MFI) on endogenous CD4 + CD44 lo , CD8 + CD44 lo , and B220 + lymphocytes ( right ) were measured in the blood of NTB or in 4T1-bearing mice treated with Iso Ab or anti-Gr-1 Ab. ( C ) Splenocytes from NTB or 4T1-bearing mice were depleted of CD11b + cells by magnetic bead isolation (94.8 ± 1.8% depletion, n = 3 mice). These cell populations were then fluorescently-labeled with different tracking dyes, co-mixed at a 1:1 ratio, and cultured in vitro or adoptively transferred into NTB recipients. Representative flow cytometric L-selectin profiles are shown for naïve CD4 + CD44 lo and CD8 + CD44 lo T cells before culture or adoptive cell transfer (ACT) (input) and four days after in culture (in vitro) or for cells recovered from blood and spleen post-ACT ( left ). Quantification of L-selectin modulation ( right ) is based on a ratio of the MFI for T cells from 4T1-bearing mice relative to NTB mice; dashed lines indicate NTB control. ( D ) MDSC or CD11b + control cells were isolated from 4T1-tumor bearing mice (tumor volume >1000 mm 3 ) or NTB mice, respectively. Myeloid cells were then co-cultured with fluorescently-labeled splenocytes from NTB mice (10:1 ratio) in media alone or with IFN-γ (20 U/mL) and LPS (100 ng/mL). MDSC and splenocytes were separated by transwell inserts (0.4 μm pore size) in the indicated co-cultures. After 24 hr, L-selectin expression on viable naive CD8 + CD44 lo T cells was analyzed by flow cytometry; representative profiles are shown ( left ). Relative changes in L-selectin expression were normalized to untreated CD8 + CD44 lo T cells (indicated by dashed lines; right ). ( A–D ) Data (mean±s.e.m.) are from one experiment ( n = 3 mice per group or ≥3 replicates per group) and are representative of ≥ two independent experiments. *p<0.05; ns, not significant; data were analyzed by unpaired two-tailed Student’s t -test. ( C , D ) Horizontal lines in histograms indicate positively stained cells; numbers are MFI. NTB, non-tumor bearing; Ab, antibody; Iso, isotype; MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.17375.014

Techniques: Control, Tumor Implantation, Expressing, Fluorescence, Isolation, Labeling, Cell Culture, In Vitro, Pore Size, Flow Cytometry, Two Tailed Test, Staining

( A ) Flow cytometric analysis of surface L-selectin (mean fluorescence intensity; MFI) on splenic naïve CD4 + CD44 lo and CD8 + CD44 lo T cells of non-tumor bearing (NTB) and 4T1-bearing mice is shown ( left ). L-selectin mRNA expression in splenic CD4 + and CD8 + T cells from NTB and 4T1-bearing mice was determined by qRT-PCR with fold-change normalized with β-actin ( right ). ( B ) Soluble (s)L-selectin in serum of individual NTB and 4T1-bearing mice was assessed by ELISA. ( A , B ) Data are from three independent experiments ( n ≥ 2 mice per group in each experiment; tumor volume >1500 mm 3 ; average frequency of splenic CD11b + Gr-1 + cells (% CD45 + leukocytes) in tumor-bearing mice was ~40%). ( C ) Splenocytes were isolated from NTB wildtype (WT) C57BL/6 mice, L(E)-selectin transgenic mice, Adam17 flox/flox /Vav1-Cre mice ( Adam17 −/ − ), or age-matched WT littermate controls. WT splenocytes were pretreated for 30 min with the ADAM17-specific inhibitor PF-5480090 (10 μM) or the ADAM17/10-specific inhibitor INCB7839 (20 μM). WT, L(E), and Adam17 − / − splenocytes were then cultured 2 hr with or without phorbol myristate acetate (PMA, 100 ng/mL). Surface L-selectin (MFI) on viable naïve CD8 + CD44 lo T cells relative to untreated controls was determined by fluorocytometric analysis. ( D ) Splenic CD11b + Gr-1 + MDSC were purified from 4T1-bearing mice; splenocytes were from various NTB mice as described in ( C ). MDSC and WT splenocytes were both pretreated for 30 min with or without PF-5480090 or INC7839. MDSC and splenocytes from WT, L(E), or Adam17 -/- mice were then co-cultured at a 10:1 ratio for 24 hr in media containing IFN-γ (20 U/mL) and LPS (100 ng/mL). L-selectin on viable naïve CD8 + CD44 lo T cells was assessed by flow cytometric analysis. ( E ) Fluorescently-labeled WT, L(E), and Adam17 − / − splenocytes (i.e., from NTB mice) were adoptively transferred into NTB severe-combined immunodeficient (SCID) mice or 4T1-bearing SCID mice at 21 days-post tumor implantation (average tumor volume for all experiments, 1102 ± 191 mm 3 ; average circulating CD11b + Gr-1 + frequencies in NTB SCID recipients, 75 ± 8 cells/µL blood, and 4T1-bearing SCID recipients, 4081 ± 876 cells/µL blood). After 24 hr post-ACT, L-selectin was assessed by flow cytometry on transferred splenocytes recovered from the blood of NTB and 4T1-bearing SCID mice. Representative flow histograms depict L-selectin expression on naïve CD8 + CD44 lo T cells ( above ); horizontal lines indicate positively stained cells, numbers are mean fluorescence intensity. Normalized data for L-selectin expression on B220 + , CD4 + CD44 lo , CD8 + CD44 lo cells 24 hr post-adoptive transfer are for one representative experiment ( n ≥ 2 mice per group) ( below ). ( A–E ) *p<0.05; ns, not significant; all data (mean±s.e.m.) were analyzed by unpaired two-tailed Student’s t -test. ( C–E ) Data are representative of ≥ two independent experiments ( n ≥ 2 replicates or mice per group) and are normalized to untreated or NTB controls (indicated by dashed lines). NTB, non-tumor bearing; WT, wildtype; MFI, mean fluorescence intensity; sL-selectin, soluble L-selectin; ACT, adoptive cell transfer. DOI: http://dx.doi.org/10.7554/eLife.17375.015

Journal: eLife

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes

doi: 10.7554/eLife.17375

Figure Lengend Snippet: ( A ) Flow cytometric analysis of surface L-selectin (mean fluorescence intensity; MFI) on splenic naïve CD4 + CD44 lo and CD8 + CD44 lo T cells of non-tumor bearing (NTB) and 4T1-bearing mice is shown ( left ). L-selectin mRNA expression in splenic CD4 + and CD8 + T cells from NTB and 4T1-bearing mice was determined by qRT-PCR with fold-change normalized with β-actin ( right ). ( B ) Soluble (s)L-selectin in serum of individual NTB and 4T1-bearing mice was assessed by ELISA. ( A , B ) Data are from three independent experiments ( n ≥ 2 mice per group in each experiment; tumor volume >1500 mm 3 ; average frequency of splenic CD11b + Gr-1 + cells (% CD45 + leukocytes) in tumor-bearing mice was ~40%). ( C ) Splenocytes were isolated from NTB wildtype (WT) C57BL/6 mice, L(E)-selectin transgenic mice, Adam17 flox/flox /Vav1-Cre mice ( Adam17 −/ − ), or age-matched WT littermate controls. WT splenocytes were pretreated for 30 min with the ADAM17-specific inhibitor PF-5480090 (10 μM) or the ADAM17/10-specific inhibitor INCB7839 (20 μM). WT, L(E), and Adam17 − / − splenocytes were then cultured 2 hr with or without phorbol myristate acetate (PMA, 100 ng/mL). Surface L-selectin (MFI) on viable naïve CD8 + CD44 lo T cells relative to untreated controls was determined by fluorocytometric analysis. ( D ) Splenic CD11b + Gr-1 + MDSC were purified from 4T1-bearing mice; splenocytes were from various NTB mice as described in ( C ). MDSC and WT splenocytes were both pretreated for 30 min with or without PF-5480090 or INC7839. MDSC and splenocytes from WT, L(E), or Adam17 -/- mice were then co-cultured at a 10:1 ratio for 24 hr in media containing IFN-γ (20 U/mL) and LPS (100 ng/mL). L-selectin on viable naïve CD8 + CD44 lo T cells was assessed by flow cytometric analysis. ( E ) Fluorescently-labeled WT, L(E), and Adam17 − / − splenocytes (i.e., from NTB mice) were adoptively transferred into NTB severe-combined immunodeficient (SCID) mice or 4T1-bearing SCID mice at 21 days-post tumor implantation (average tumor volume for all experiments, 1102 ± 191 mm 3 ; average circulating CD11b + Gr-1 + frequencies in NTB SCID recipients, 75 ± 8 cells/µL blood, and 4T1-bearing SCID recipients, 4081 ± 876 cells/µL blood). After 24 hr post-ACT, L-selectin was assessed by flow cytometry on transferred splenocytes recovered from the blood of NTB and 4T1-bearing SCID mice. Representative flow histograms depict L-selectin expression on naïve CD8 + CD44 lo T cells ( above ); horizontal lines indicate positively stained cells, numbers are mean fluorescence intensity. Normalized data for L-selectin expression on B220 + , CD4 + CD44 lo , CD8 + CD44 lo cells 24 hr post-adoptive transfer are for one representative experiment ( n ≥ 2 mice per group) ( below ). ( A–E ) *p<0.05; ns, not significant; all data (mean±s.e.m.) were analyzed by unpaired two-tailed Student’s t -test. ( C–E ) Data are representative of ≥ two independent experiments ( n ≥ 2 replicates or mice per group) and are normalized to untreated or NTB controls (indicated by dashed lines). NTB, non-tumor bearing; WT, wildtype; MFI, mean fluorescence intensity; sL-selectin, soluble L-selectin; ACT, adoptive cell transfer. DOI: http://dx.doi.org/10.7554/eLife.17375.015

Techniques: Fluorescence, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Isolation, Transgenic Assay, Cell Culture, Purification, Labeling, Tumor Implantation, Flow Cytometry, Staining, Adoptive Transfer Assay, Two Tailed Test

( A ) Schematic of competitive activation assays. CD8 + T cell populations (>95% CD8 + ) were isolated from non-tumor bearing OT-I mice (NTB OT-I CD8 + L-selectin hi ) and from AT-3-bearing OT-1 mice (AT-3 OT-I CD8 + L-selectin intermediate-to-low; L-selectin int/lo ; tumor volume 3825 ± 123 mm 3 for n = 4 mice). T cells (depleted of CD11b + MDSC) from NTB or tumor-bearing mice were then labeled ex vivo with different proliferation dyes (CellTrace Violet or CellTrace CFSE, respectively), co-mixed at a 1:1 ratio, and assessed for functional responses to cognate antigen (SIINFEKL) after four days in competitive activation assays in vitro and in vivo. ( B ) Flow cytometric analysis of proliferation of NTB OT-I CD8 + and AT-3 OT-I CD8 + T cells after activation by SIINFEKL-loaded dendritic cells (DC) for four days in vitro. Horizontal lines on histograms indicate percent proliferating cells. ( C ) Competitive in vivo activation assay in which NTB recipient mice were vaccinated (via footpad) with SIINFEKL-loaded DC 6 hr before adoptive transfer of a 1:1 mixture of L-selectin hi NTB OT-I and L-selectin int/lo AT-3 OT-I CD8 + T cells. After four days, the ratios of the adoptively transferred cells were assessed in the following lymphoid compartments: spleen (Spl), contralateral popliteal lymph node (cLN), and draining popliteal lymph node (dLN). Data (mean±s.e.m.) are from one experiment ( n = 4 mice per group) and are representative of 2 independent experiments. *p<0.05; ns, not significant; data were analyzed by unpaired two-tailed Student’s t -test. ( D ) IFN-γ expression profiles for endogenous CD8 + T cells and adoptively transferred NTB OT-I CD8 + and AT-3 OT-I CD8 + T cells recovered in dLN of DC-vaccinated mice. Horizontal lines on histograms indicate positively stained cells; data are representative of 2 mice per group. NTB, non-tumor bearing; DC, dendritic cell; Iso, Isotype; Ab, antibody. DOI: http://dx.doi.org/10.7554/eLife.17375.023

Journal: eLife

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes

doi: 10.7554/eLife.17375

Figure Lengend Snippet: ( A ) Schematic of competitive activation assays. CD8 + T cell populations (>95% CD8 + ) were isolated from non-tumor bearing OT-I mice (NTB OT-I CD8 + L-selectin hi ) and from AT-3-bearing OT-1 mice (AT-3 OT-I CD8 + L-selectin intermediate-to-low; L-selectin int/lo ; tumor volume 3825 ± 123 mm 3 for n = 4 mice). T cells (depleted of CD11b + MDSC) from NTB or tumor-bearing mice were then labeled ex vivo with different proliferation dyes (CellTrace Violet or CellTrace CFSE, respectively), co-mixed at a 1:1 ratio, and assessed for functional responses to cognate antigen (SIINFEKL) after four days in competitive activation assays in vitro and in vivo. ( B ) Flow cytometric analysis of proliferation of NTB OT-I CD8 + and AT-3 OT-I CD8 + T cells after activation by SIINFEKL-loaded dendritic cells (DC) for four days in vitro. Horizontal lines on histograms indicate percent proliferating cells. ( C ) Competitive in vivo activation assay in which NTB recipient mice were vaccinated (via footpad) with SIINFEKL-loaded DC 6 hr before adoptive transfer of a 1:1 mixture of L-selectin hi NTB OT-I and L-selectin int/lo AT-3 OT-I CD8 + T cells. After four days, the ratios of the adoptively transferred cells were assessed in the following lymphoid compartments: spleen (Spl), contralateral popliteal lymph node (cLN), and draining popliteal lymph node (dLN). Data (mean±s.e.m.) are from one experiment ( n = 4 mice per group) and are representative of 2 independent experiments. *p<0.05; ns, not significant; data were analyzed by unpaired two-tailed Student’s t -test. ( D ) IFN-γ expression profiles for endogenous CD8 + T cells and adoptively transferred NTB OT-I CD8 + and AT-3 OT-I CD8 + T cells recovered in dLN of DC-vaccinated mice. Horizontal lines on histograms indicate positively stained cells; data are representative of 2 mice per group. NTB, non-tumor bearing; DC, dendritic cell; Iso, Isotype; Ab, antibody. DOI: http://dx.doi.org/10.7554/eLife.17375.023

Techniques: Activation Assay, Isolation, Labeling, Ex Vivo, Functional Assay, In Vitro, In Vivo, Adoptive Transfer Assay, Two Tailed Test, Expressing, Staining

( A ) Schematic of MDSC preconditioning protocol. MDSC were isolated from the blood of 4T1-bearing mice (≥93% CD11b + Gr-1 + ) and co-cultured with splenocytes from non-tumor bearing (NTB) OT-I and DO11.10 T cell receptor (TcR) transgenic mice. After 16 hr, MDSC were depleted by magnetic separation using biotinylated anti-Gr-1 antibody and streptavidin beads. Remaining splenocyte populations contained 0.4–4% CD11b + Gr-1 + cells. Cognate peptide was then added to each culture. After three days in culture, splenocytes were pulsed with 3 H-thymidine and harvested 16 hr later. ( B ) Proliferative responses are shown for OT-I CD8 + ( left ) and DO11.10 CD4 + ( right ) T cells based on 3 H-thymidine incorporation (CPM). Ratios denote the relative proportion of purified MDSC:OT-I or MDSC:DO11.10 splenocytes used in the preconditioning phase. Data (mean±s.d.) are of six replicate cultures in a single experiment and are representative of three independent experiments. *p<0.002; unpaired two-tailed Student’s t -test. CPM, counts per minute; TcR, T cell receptor; NTB, non-tumor bearing. DOI: http://dx.doi.org/10.7554/eLife.17375.024

Journal: eLife

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes

doi: 10.7554/eLife.17375

Figure Lengend Snippet: ( A ) Schematic of MDSC preconditioning protocol. MDSC were isolated from the blood of 4T1-bearing mice (≥93% CD11b + Gr-1 + ) and co-cultured with splenocytes from non-tumor bearing (NTB) OT-I and DO11.10 T cell receptor (TcR) transgenic mice. After 16 hr, MDSC were depleted by magnetic separation using biotinylated anti-Gr-1 antibody and streptavidin beads. Remaining splenocyte populations contained 0.4–4% CD11b + Gr-1 + cells. Cognate peptide was then added to each culture. After three days in culture, splenocytes were pulsed with 3 H-thymidine and harvested 16 hr later. ( B ) Proliferative responses are shown for OT-I CD8 + ( left ) and DO11.10 CD4 + ( right ) T cells based on 3 H-thymidine incorporation (CPM). Ratios denote the relative proportion of purified MDSC:OT-I or MDSC:DO11.10 splenocytes used in the preconditioning phase. Data (mean±s.d.) are of six replicate cultures in a single experiment and are representative of three independent experiments. *p<0.002; unpaired two-tailed Student’s t -test. CPM, counts per minute; TcR, T cell receptor; NTB, non-tumor bearing. DOI: http://dx.doi.org/10.7554/eLife.17375.024

Techniques: Isolation, Cell Culture, Transgenic Assay, Purification, Two Tailed Test

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