Journal: Cancer immunology research

Article Title: TLR5 signaling causes dendritic-cell dysfunction and orchestrates failure of immune checkpoint therapy against ovarian cancer

doi: 10.1158/2326-6066.CIR-24-0513

Figure Lengend Snippet: Tumors were initiated in wild-type (WT), TLR5 KO or TLR4 KO mice as indicated. After 10 days of tumor progression, mice were given either anti-PD-L1 or anti-CTLA4 every 3–4 days for a total of 4 injections. Survival of mice bearing ID8- Defb29/Vegf-A ( A ), UPK10 ( B ), or PPNM ( C ) ovarian tumors after treatment with anti-PD-L1. ( D ) Survival of TLR4 KO or wild type controls bearing ID8- Defb29/Vegf-A ovarian tumors after treatment with anti-PD-L1. ( E ) Survival of mice bearing ID8- Defb29/Vegf-A ovarian tumors after treatment with anti-CTLA4. A log-rank test was used to compare survival proportions. Numbers in parentheses are number of mice/group with a total of at least 2 independent experiments. (* p < .05, ** p < .01, *** p < .001, **** p < .0001).

Article Snippet: TLR5 TdTomato reporter mice (TLR5KI-tdTom - B6(FVB)- Tlr5 tm1.1 Gbrt/J )( 22 ), TLR4KO mice (B6(Cg)- Tlr4 tm1.2Karp /J) and matching wild-type C57BL/6 controls were also from The Jackson Laboratory.

Techniques:

Journal: Cancer immunology research

Article Title: TLR5 signaling causes dendritic-cell dysfunction and orchestrates failure of immune checkpoint therapy against ovarian cancer

doi: 10.1158/2326-6066.CIR-24-0513

Figure Lengend Snippet: ( A ) Peritoneal wash exudates from TdTomato TLR5 reporter mice bearing ID8- Defb29/Vegf-A ovarian tumors were analyzed by flow at day 25 for TLR5-expressing (TdTomato + ) cells in the TME. Percentages based upon frequency of TLR5 + or TLR5 − cells out of total CD45 + . N=14 total mice. ( B - C ) Analysis of TLR5 expressing cell subsets in the peritoneum of naïve mice (no tumor) and in the TME of mice with ovarian tumors at 10-, 20-, and 30-days post-tumor. N=5 mice per group. Percentages are based upon total TLR5 + or TLR5 − cell proportions expressing each set of surface markers defined in the legend and depicted as stacked bar graphs ( B ) or pie graphs ( C ). Phenotypic analysis of TLR5 + ( D and F ) or TLR5 − ( E and G ) MHCII hi and CD11c + DC cell subsets of TdTomato TLR5 reporter mice. ( D and E ) proportions of each phenotypic DC subset from TLR5 + or TLR5 − cells. ( F and G ) MFI of each marker from cells in D and E . N=5 per group. For all p values, Mann-Whitney unpaired t-test (* p < .05, ** p < .01, *** p < .001, **** p < .0001) was used to calculate significance. Error bars represent mean ± SEM. All graphs are representative of at least 2 individual experiments.

Article Snippet: TLR5 TdTomato reporter mice (TLR5KI-tdTom - B6(FVB)- Tlr5 tm1.1 Gbrt/J )( 22 ), TLR4KO mice (B6(Cg)- Tlr4 tm1.2Karp /J) and matching wild-type C57BL/6 controls were also from The Jackson Laboratory.

Techniques: Expressing, Marker, MANN-WHITNEY

Journal: Cancer immunology research

Article Title: TLR5 signaling causes dendritic-cell dysfunction and orchestrates failure of immune checkpoint therapy against ovarian cancer

doi: 10.1158/2326-6066.CIR-24-0513

Figure Lengend Snippet: A-D: Dendritic cells in the peritoneal environment of wild-type and TLR5 KO mice were analyzed in the absence of a tumor (non-tumor-bearing) or 7- and 15-days post initiation of ID8 -Defb29/Vegf-A ovarian tumors. N = 4 mice/group. Plots representative of at least 3 individual experiments. ( A ) Total cDC1 and ( B ) IL-12+ cDC1 in the peritoneal cavity. ( C ) MFI ( D ) and representative histograms of intracellular IL-12 and surface CD80 levels in cDC1s from the ovarian TME 15 days post ID8 -Defb29/Vegf-A tumors. ( E ) Schematic depicting strategy to neutralize TLR5 signaling. Peritoneal wash samples and tumor nodules were analyzed on day 30 post-tumor. N = 4 mice/group. Plots are representative of at least 3 individual experiments. ( F ) t-SNE map of pre-gated CD11c+ MHCII+ cells. Red circle depicts cDC1 populations based upon CD103 and XCR1 expression, highlighted by the arrow in G and H . ( G ) Population frequencies and ( H ) heat map of phenotypic marker expression by DCs from the clusters in F . ( I ) Quantitation of cDC1 numbers and ( J ) MFI of PD-L1 levels with representative histograms. Unpaired non-parametric t-tests with Mann-Whitney correction was used to calculate statistical significance for all plots (* p < .05, ** p < .01, *** p < .001, **** p < .0001). Error bars represent mean ± SEM.

Article Snippet: TLR5 TdTomato reporter mice (TLR5KI-tdTom - B6(FVB)- Tlr5 tm1.1 Gbrt/J )( 22 ), TLR4KO mice (B6(Cg)- Tlr4 tm1.2Karp /J) and matching wild-type C57BL/6 controls were also from The Jackson Laboratory.

Techniques: Expressing, Marker, Quantitation Assay, MANN-WHITNEY

Journal: Cancer immunology research

Article Title: TLR5 signaling causes dendritic-cell dysfunction and orchestrates failure of immune checkpoint therapy against ovarian cancer

doi: 10.1158/2326-6066.CIR-24-0513

Figure Lengend Snippet: ( A ) Schema for mixed bone marrow chimera and treatment regimen. Briefly, wild-type CD45.1 congenic mice were lethally irradiated, followed by transfer of a 50:50 mixture of wild-type (CD45.1) and TLR5 KO (CD45.2) bone marrow. After 10 weeks, ID8 -Defb29/Vegf-A tumors were initiated, followed by the initiation of PD-L1 blockade. After 15 days post-tumor, peritoneal wash exudates/tumor nodules were analyzed using flow cytometry. ( B ) Frequency of wild-type (CD45.1) or TLR5 KO (CD45.2) XCR1 + CD103 + (cDC1), ( C ) CD11b + CD103 + (mucosal DC), ( D ) CD11b + CD103 − (myeloid DC), ( E ) PD-L1hi cDC1 (XCR1 + CD103 + ) with or without anti-PD-L1 therapy. ( F ) Frequency of wild-type (CD45.1) or TLR5 KO (CD45.2) IL-12 + cDC1s. Unpaired Mann-Whitney (between mice) or paired t-tests (within mice) were used to calculate significance (* p < .05, ** p < .01, *** p < .001, **** p < .0001). Dots represent individual animals. Plots are representative of three independent experiments, with 5–10 mice/group.

Article Snippet: TLR5 TdTomato reporter mice (TLR5KI-tdTom - B6(FVB)- Tlr5 tm1.1 Gbrt/J )( 22 ), TLR4KO mice (B6(Cg)- Tlr4 tm1.2Karp /J) and matching wild-type C57BL/6 controls were also from The Jackson Laboratory.

Techniques: In Vivo, Irradiation, Flow Cytometry, MANN-WHITNEY

Journal: Cancer immunology research

Article Title: TLR5 signaling causes dendritic-cell dysfunction and orchestrates failure of immune checkpoint therapy against ovarian cancer

doi: 10.1158/2326-6066.CIR-24-0513

Figure Lengend Snippet: Bone marrow from wild-type or TLR5 KO mice were cultured for 8 days with FLT3L and purified flagellin ( Salmonella typhimurium ) for 2 days (2D - acute) or 8 days (8D - chronic) of culture. ( A ) Total numbers of differentiated myeloid and dendritic cell subsets: cDC1s (CD11c + , MHCII hi , XCR1 + , SIRPα − ), cDC2s (CD11c + , MHCII hi , XCR1 − , SIRPα + ), Ly6C (CD11c − , CD11b + , Ly6C + , Ly6G − ), Ly6G (CD11c − , CD11b + , Ly6C low , Ly6G + ), and other undefined myeloid cells (CD11c − , CD11b + , Ly6C − , Ly6G − ). ( B ) Total numbers of PD-L1 high expressing subsets from A . ( C-D ) Bulk DC/myeloid cultures were pulsed with SIINFEKL peptide followed by incubation with OT-1 transgenic T cells, followed by analysis of T cell proliferation. ( C ) Proliferation index and ( D ) representative histograms of cell trace violet-labeled proliferating OT-1 CD8 T cells. Unpaired t-test with Mann-Whitney correction was used to calculate significance. (* p < .05, ** p < .01, *** p < .001, **** p < .0001). Each point represents cultures from an individual animal. Plots represent all data points combined from two experiments. ( E-G ) CITE-seq was performed on FLT3L bone marrow cultures with or without chronic flagellin stimulation for 8 days. ( E ) Gene set enrichment analysis between cultures expanded in the presence of chronic flagellin stimulation versus cultures expanded in the presence of FLT3L only. ( F ) Volcano plot of differentially expressed genes for cDC1 cells (pseudobulk analysis was performed on cells in cluster 10, identified cDC1s, grouped according to their origin). ( G ) Pathway analysis of differentially expressed genes identified in cluster 10 by grouping cells to their origin.

Article Snippet: TLR5 TdTomato reporter mice (TLR5KI-tdTom - B6(FVB)- Tlr5 tm1.1 Gbrt/J )( 22 ), TLR4KO mice (B6(Cg)- Tlr4 tm1.2Karp /J) and matching wild-type C57BL/6 controls were also from The Jackson Laboratory.

Techniques: Cell Culture, Purification, Expressing, Incubation, Transgenic Assay, Labeling, MANN-WHITNEY

Journal: Cancer immunology research

Article Title: TLR5 signaling causes dendritic-cell dysfunction and orchestrates failure of immune checkpoint therapy against ovarian cancer

doi: 10.1158/2326-6066.CIR-24-0513

Figure Lengend Snippet: ( A ) Treatment schema with IP administration of anti-CD8a (αCD8a) and anti-PD-L1. Briefly, wild-type or TLR5 KO mice were treated with αCD8a prior to and after tumor initiation with ID8 -Defb29/Vegf-A tumors and PD-L1 blockade. ( B ) Survival proportions of TLR5 KO and wild-type mice bearing ID8 -Defb29/Vegf-A tumors. Log-rank test for survival compared to wild-type (* p < .05, ** p < .01, *** p < .001, **** p < .0001 ) N=5 per group and is representative of two independent experiments. ( C ) Treatment schema for rechallenge of TLR5 KO mice surviving ID8 -Defb29/Vegf-A or UPK10 tumors after PD-L1 blockade ( from 1A and B ). After 100 days, surviving or naïve TLR5 KO mice were injected with the same ovarian tumor cell line as established in surviving mice on day 0 with survival being monitored in the absence of PD-L1 blockade. Survival of rechallenged TLR5 KO mice with ID8- Defb29/Vegf-A tumors ( D ) or UPK10 tumors ( E ). Log-rank test was used to compare survival proportions. Numbers in parentheses indicate total mice within each group combining at least 2 independent experiments. (* p < .05, ** p < .01, *** p < .001, **** p < .0001). ( F-H ) (F) Treatment schema to investigate T cell function during acute (7 days post-tumor, +7D) or 7 days after tumor rechallenge of TLR5 KO mice surviving a primary or secondary challenge with ID8 -Defb29/Vegf-A tumors. Secondary challenge indicates surviving TLR5 KO mice that received a re-injection of ID8 -Defb29/Vegf-A tumors 100 days after surviving primary tumor challenge. Primary challenge indicates surviving TLR5 KO mice after primary tumor challenge and treatment with PD-L1 blockade. All mice were assessed 7 days following ID8 -Defb29/Vegf-A tumor administration. ( G ) Representative gating of CD8 T cells expressing CD107a and IFNγ. ( H ) Quantification of T cells within the ovarian TME. Unpaired t-test with Mann-Whitney Correction (* p < .05, ** p < .01, *** p < .001, **** p < .0001 ). Error bars represent mean ± SEM. Each dot represents an individual animal. Plots are representative of two experiments with an 4–5 mice/group.

Article Snippet: TLR5 TdTomato reporter mice (TLR5KI-tdTom - B6(FVB)- Tlr5 tm1.1 Gbrt/J )( 22 ), TLR4KO mice (B6(Cg)- Tlr4 tm1.2Karp /J) and matching wild-type C57BL/6 controls were also from The Jackson Laboratory.

Techniques: Injection, Cell Function Assay, Expressing, MANN-WHITNEY

Journal: Cancer immunology research

Article Title: TLR5 signaling causes dendritic-cell dysfunction and orchestrates failure of immune checkpoint therapy against ovarian cancer

doi: 10.1158/2326-6066.CIR-24-0513

Figure Lengend Snippet: A-I TLR5fl/fl × CD11c cre negative (CD11c.TLR5 wt .cre neg ), CD11c Cre positive (CD11c.TLR5 wt .cre), and TLR5fl/fl × CD11c Cre positive (CD11c.TLR5 ko .cre) mice were given ID8 -Defb29/Vegf-A ovarian tumors, followed by administration of PD-L1 blockade 10 days post-tumor-initiation. ( A ) Survival proportions of CD11c.TLR5 wt .cre neg , CD11c.TLR5 wt .cre, and CD11c.TLR5 ko .cre mice. Log-rank test for survival compared to CD11c.TLR5 wt .cre neg and CD11c.TLR5 wt .cre strains (* p < .05, ** p < .01, *** p < .001, **** p < .0001). Numbers in parenthesis indicate total number of mice and encompass two independent experiments. ( B-I ) 25 days post-initiation of ID8 -Defb29/Vegf-A tumors in CD11c.TLR5 wt (cre negative) and CD11c.TLR5 ko mice (cre positive), peritoneal wash exudates and tumor nodules were evaluated using flow cytometry. ( B ) Total numbers of cDC1s, ( C ) PD-L1hi cDC1s, ( D ) MFI of PD-L1 and ( E ) representative histograms of PD-L1 on CD11c + MHCII + cells. ( F ) Total PD-1 + Lag3 + CD8 T cells in the TME. ( G ) Representative gating of CD3 + CD44hi CD107 + or IFNγ + T cells. ( H ) Total CD3 + CD107a + and ( I ) CD3 + IFNγ + . Unpaired t-test with Mann-Whitney correction was used to calculate significance (* p < .05, ** p < .01, *** p < .001, **** p < .0001). Error bars represent mean ± SEM. Dot plots are representative of at least three experiments, with each dot representing an individual animal. ( J-K ) Survival proportions of TLR5 KO and wild-type mice bearing ID8- Defb29/Vegf-A tumors ( J ) or PPNM tumors ( K ) and treated with FLT3L and/or anti-PD-L1. Log-rank test for survival compared to wild type (* p < .05, ** p < .01, *** p < .001, **** p < .0001). N=5 per group, results representative of at least two independent experiments.

Article Snippet: TLR5 TdTomato reporter mice (TLR5KI-tdTom - B6(FVB)- Tlr5 tm1.1 Gbrt/J )( 22 ), TLR4KO mice (B6(Cg)- Tlr4 tm1.2Karp /J) and matching wild-type C57BL/6 controls were also from The Jackson Laboratory.

Techniques: Cell Function Assay, Flow Cytometry, MANN-WHITNEY