Journal: Nature

Article Title: PGE 2 limits effector expansion of tumour-infiltrating stem-like CD8 + T cells

doi: 10.1038/s41586-024-07254-x

Figure Lengend Snippet: a , Experimental design for b – f . b , Flow cytometric plots of CD8 + T cells from tdLNs and tumours from the indicated days. c , d , Numbers of expanded OT-I CD8 + T cells in tdLNs ( c ) and tumours ( d ) at indicated time points ( n = 6). e , f , Analysis of CD44 and CXCR6 expression in Cd4 cre Ptger2 −/− Ptger4 fl/fl OT-I cells. e , Flow cytometry plots. f , Subset frequencies ( n = 6). g – j , Effect of CD122/CD132 blockade on OT-I T cell expansion in tumours. g – j , Effect of anti-CD122 and anti-CD132 (anti-CD122/CD132) treatment on OT-I TIL expansion in WT mice with control or Ptgs1/Ptgs2 −/− BRAF V600E -OVA tumours or with MC38-OVA tumours, analysed 11 days after tumour transplantation. g , h , Flow cytometry plots ( g ) and OT-I TIL numbers ( h ) in BRAF V600E -OVA tumours ( n = 6). i , j , Flow cytometry plots ( i ) and OT-I TIL numbers ( j ) in MC38-OVA tumours ( n = 10). k , Experimental design for l and m with MC38-OVA tumours. l , Flow cytometry plot (left) and quantification (right) of OT-I TILs at day 10 ( n = 6). m , Flow cytometry plots showing the population size of TIM-3 + CXCR6 + cells among control and Cd122 −/− Cd4 cre Ptger2 −/− Ptger4 fl/fl OT-I TILs. n , WT mice received 1 × 10 3 naive OT-I T cells or 1 × 10 3 naive Cd4 c re Ptger2 −/− Ptger4 fl/fl OT-I T cells intravenously (i.v.) and were transplanted s.c. with 2 × 10 6 MC38-OVA cells before analysis of tumour growth over time ( n = 10). Asterisk indicates that termination criteria were reached. Data in c , d , f , h , j , l and n are pooled from two ( c , d , h , l ) or three ( f , j , n ) independent experiments and depicted as box plots extending from the 25th to 75th percentiles with the median as the centre and the whiskers corresponding to minimum and maximum values ( c , d , h , j , l ) or shown as the mean ± s.e.m. ( f , n ). Plots in b , e , g , i , l and m show data for 1 sample representative of n = 6 samples analysed in 2 ( b , g , l , m ) or 3 ( e , i ) independent experiments. P values are from paired t -tests ( l ), one-way ANOVA with Tukey’s multiple-comparison test ( c , d ) or Dunnett’s multiple-comparison test ( h , j ), or two-way ANOVA with Bonferroni’s correction for multiple testing ( n ).

Article Snippet: For blockade of IL-2Rβ and IL-2Rγc, mice received i.p. injections of 150 µl anti-mouse CD122 (300 µg per mouse, TM-Beta 1, BioXCell) and anti-mouse CD132 (300 µg per mouse, 3E12, BioXCell) antibodies on days 6 and 7 after tumour cell transplantation.

Techniques: Expressing, Flow Cytometry, Transplantation Assay, Comparison

Journal: Frontiers in Immunology

Article Title: A New Immunosuppressive Molecule Emodin Induces both CD4 + FoxP3 + and CD8 + CD122 + Regulatory T Cells and Suppresses Murine Allograft Rejection

doi: 10.3389/fimmu.2017.01519

Figure Lengend Snippet: Emodin suppresses the expansion of effector CD8 + T cells. (A) Draining lymph node (LN) and spleen cells from emodin- and CsA-treated B6 mice transplanted with BALB/c skin were isolated 10 days after transplantation and analyzed via FACS analysis. Column graphs show the percentages of CD8 + CD44 high CD62L low effector T cells (Teff) from LNs and spleens. (B) The proliferation of CD8 + CD44 high CD62L low T cells was analyzed by EDU labeling. Recipient mice were pulsed intraperitoneally with EDU 10 days after transplantation. 24 h later, LN and spleen cells were harvested and stained for CD8, CD44, CD62L, and EDU. Histograms are gated on CD8 + CD44 high CD62L low population. The percentage of EDU-positive CD8 + CD44 high CD62L low cells also is shown in column graphs. The apoptosis of CD8 + CD44 high CD62L low (C) and CD4 + CD44 high CD62L low (D) Teff cells was measured by annexin V labeling. LN and spleen cells were stained for CD8, CD4, CD44, CD62L, and annexin V and analyzed by flow cytometry. The percentage of Annexin V-positive CD8 + CD44 high CD62L low and CD4 + CD44 high CD62L low Teff cells is also shown in column graphs. Data are presented as means ± SD from two separate experiments (* P < 0.05, ** P < 0.01, n = 4 mice/group). One representative of three separate experiments is shown for all panels.

Article Snippet: To purify CD4 + CD25 + and CD8 + CD122 + Tregs for adoptive transfer experiments, spleen cells were stained with anti-CD4-PE (Clone RM4-5) and anti-CD25-FITC (Clone 3C7) or, separately, anti-CD8-PE (Clone 53-6.7) and anti-CD122-FITC (Clone TM-Beta 1) Abs (BD Biosciences).

Techniques: Isolation, Transplantation Assay, Labeling, Staining, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: A New Immunosuppressive Molecule Emodin Induces both CD4 + FoxP3 + and CD8 + CD122 + Regulatory T Cells and Suppresses Murine Allograft Rejection

doi: 10.3389/fimmu.2017.01519

Figure Lengend Snippet: Emodin also augments the percentages of CD8 + CD122 + Tregs. Draining LN and spleen cells from emodin- or CsA-treated B6 mice transplanted with BALB/c skin were isolated 10 days after transplantation. The percentages of CD8 + CD122 + Tregs from LNs and spleens of recipient mice were measured via a flow cytometer. Data are shown as means ± SD from two separate experiments (* P < 0.05 and ** P < 0.01, n = 4–5 mice/group).

Article Snippet: To purify CD4 + CD25 + and CD8 + CD122 + Tregs for adoptive transfer experiments, spleen cells were stained with anti-CD4-PE (Clone RM4-5) and anti-CD25-FITC (Clone 3C7) or, separately, anti-CD8-PE (Clone 53-6.7) and anti-CD122-FITC (Clone TM-Beta 1) Abs (BD Biosciences).

Techniques: Isolation, Transplantation Assay, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: A New Immunosuppressive Molecule Emodin Induces both CD4 + FoxP3 + and CD8 + CD122 + Regulatory T Cells and Suppresses Murine Allograft Rejection

doi: 10.3389/fimmu.2017.01519

Figure Lengend Snippet: Emodin-induced CD4 + CD25 + or CD8 + CD122 + regulatory T cells (Tregs) suppress skin allograft rejection in Rag1 −/− recipient mice. CD4 + CD25 + or CD8 + CD122 + Tregs were isolated via FACS cell sorting from B6 recipient mice that were transplanted with BALB/c skin and treated with emodin and/or CsA. CD4 + CD25 + (A) or CD8 + CD122 + (B) Tregs (0.4 × 10 6 ), together with B6-derived naïve CD3 + T cells (2 × 10 6 ), were adoptively transferred to Rag1 −/− mice (B6 background) that were then transplanted with a BALB/c skin graft. Skin allograft rejection in Rag1 −/− recipient mice was observed ( n = 7–8 grafts).

Article Snippet: To purify CD4 + CD25 + and CD8 + CD122 + Tregs for adoptive transfer experiments, spleen cells were stained with anti-CD4-PE (Clone RM4-5) and anti-CD25-FITC (Clone 3C7) or, separately, anti-CD8-PE (Clone 53-6.7) and anti-CD122-FITC (Clone TM-Beta 1) Abs (BD Biosciences).

Techniques: Isolation, FACS, Derivative Assay

Journal: Cancer immunology research

Article Title: Sustained persistence of IL2 signaling enhances the antitumor effect of peptide vaccines through T-cell expansion and preventing PD-1 inhibition

doi: 10.1158/2326-6066.CIR-17-0549

Figure Lengend Snippet: Irrelevant CD8+ T cells inhibition of antigen-specific CD8+ T-cell responses to peptide vaccines is reversed by IL2Cx. A, WT mice were injected with Trp1-BiVax or Trp1-Trivax. One week later, the total numbers of antigen-specific (tetramer+) and tetramer− CD8+ T cells were evaluated in spleens. B, WT or RAG1-KO mice were adoptively transferred with CD8+ T cells from TgTR1 mice (2000 cells/mouse) with or without WT CD8+ T cells (8 × 106/mouse). One and 12 days later, mice were vaccinated with Trp1-BiVax. On day 19, the total numbers of TgTR1 cells were evaluated in spleens. C, Pmel-1 mice (Thy1.1+, RAG+) received 2000 TgTR1 cells (Thy1.2+) followed by two Trp1-BiVax vaccinations 12 days apart. In some mice Thy1.1+ cells (all T cells) or CD4+ T cells were depleted using mAb and one group of mice was injected with IL2Cx122 on days 12, 14 and 16. The total numbers of TgTR1 cells (Tetramer+ T cells) were evaluated in spleens on day 19. D, WT mice were vaccinated as shown and the percentage of Trp1 tetramer+ T cells was evaluated in the blood 7 days after each vaccination. E, Total numbers of tetramer+ T cells in spleen, 8 days after the boost. F, Purified spleen CD8+ T cells (105) were incubated with B16F10 cells overnight and the numbers of IFNγ producing cells were evaluated using EliSpot. Representative results are shown from at least 3 independent experiments (n = 3 mice/group).

Article Snippet: Poly-ICLC was provided by Dr. Andres Salazar (Oncovir). mAb against mouse CD40 (FGK4.5, cat. # BE0016-2), mouse PD-L1 (10F.9G2, cat. # BE0101), mouse IL2 (JES6-5H4, cat. # BE0042 JES6-1A12, cat. # BE0043), mouse CD4 (GK1.5, cat. # BE0003-1), mouse CD122 (TM-beta 1, cat # BE0298), mouse CD25 (PC-61.5.3, cat # BE00012) and mouse Thy1.1 (19E12, cat. # BE0214) were purchased from BioXcell.

Techniques: Inhibition, Injection, Purification, Incubation, Enzyme-linked Immunospot

Journal: Cancer immunology research

Article Title: Sustained persistence of IL2 signaling enhances the antitumor effect of peptide vaccines through T-cell expansion and preventing PD-1 inhibition

doi: 10.1158/2326-6066.CIR-17-0549

Figure Lengend Snippet: Sustained IL2 stimulation is responsible for the adjuvant effect of IL2Cx. A, TgTR1 cells were incubated with Trp1 peptide (1 µg/ml) and IL2Cx122 or IL2Cx25 (100 ng IL2/ml) in the presence of increasing concentrations of homologue IL2 mAb (JES6-5H4 or JES6-1A12, respectively). Seven days later T-cell expansion was evaluated. B, CD45.1 mice were adoptively transferred with 1 × 105 TgTR1 cells followed by BiVax prime. Five days later the mice were boosted with BiVax, BiVax/IL2Cx25(10 µg) (2 µg IL2 + 10 µg IL2 mAb) or BiVax/IL2Cx25(300 µg) (2 µg IL2 + 300 µg IL2 mAb). On day 12 the total numbers of TgTR1 cells were evaluated in spleens. C, Representative flow dot plots from the experiment in panel B, showing the percentage of TgTR1 cells (MHCII-CD45.2+) in spleen gating in total live cells. (n = 3 mice/group). Representative results are shown from at least 3 independent experiments.

Article Snippet: Poly-ICLC was provided by Dr. Andres Salazar (Oncovir). mAb against mouse CD40 (FGK4.5, cat. # BE0016-2), mouse PD-L1 (10F.9G2, cat. # BE0101), mouse IL2 (JES6-5H4, cat. # BE0042 JES6-1A12, cat. # BE0043), mouse CD4 (GK1.5, cat. # BE0003-1), mouse CD122 (TM-beta 1, cat # BE0298), mouse CD25 (PC-61.5.3, cat # BE00012) and mouse Thy1.1 (19E12, cat. # BE0214) were purchased from BioXcell.

Techniques: Incubation

Journal: Cancer immunology research

Article Title: Sustained persistence of IL2 signaling enhances the antitumor effect of peptide vaccines through T-cell expansion and preventing PD-1 inhibition

doi: 10.1158/2326-6066.CIR-17-0549

Figure Lengend Snippet: PEG-IL2 is effective in expanding BiVax generated antigen-specific T cells. A, WT mice (3/group) were vaccinated with BiVax prime. Five days later the mice received boosters with BiVax, BiVax/IL2, BiVax/IL2Cx122 or BiVax/PEG-IL2. Seven days later the numbers of Trp1 tetramer+ T cells were evaluated in spleens. B, WT mice were inoculated s.c. with B16F10 cells and 7 days later, received a BiVax prime followed 5 days later by booster vaccines with BiVax, BiVax/IL2 or BiVax/PEG-IL2. Tumor size growth curves showing means with SD for each group. (n = 10 mice/group). C, purified CD8+ T cells from WT mice receiving BiVax prime-boost (5 days apart) and treated or not with PEG-IL2 were incubated with either B16F10 (PD-L1 low) cells, IFNγ-treated (PD-L1 high) B16F10 cells (pulsed with 1 µg Trp1) with or without PD-L1 mAb (10 µg/ml) and the numbers of IFNγ-producing cells were evaluated by EliSpot. Representative results are shown from at least 3 independent experiments.

Article Snippet: Poly-ICLC was provided by Dr. Andres Salazar (Oncovir). mAb against mouse CD40 (FGK4.5, cat. # BE0016-2), mouse PD-L1 (10F.9G2, cat. # BE0101), mouse IL2 (JES6-5H4, cat. # BE0042 JES6-1A12, cat. # BE0043), mouse CD4 (GK1.5, cat. # BE0003-1), mouse CD122 (TM-beta 1, cat # BE0298), mouse CD25 (PC-61.5.3, cat # BE00012) and mouse Thy1.1 (19E12, cat. # BE0214) were purchased from BioXcell.

Techniques: Generated, Purification, Incubation, Enzyme-linked Immunospot

Journal: Cancer immunology research

Article Title: Sustained persistence of IL2 signaling enhances the antitumor effect of peptide vaccines through T-cell expansion and preventing PD-1 inhibition

doi: 10.1158/2326-6066.CIR-17-0549

Figure Lengend Snippet: Comparisons of BiVax/IL2Cx and TriVax for immunogenicity and antitumor efficacy. A, CD45.1 WT mice received 1 × 105 TgTR1 T cells (CD45.2) and were vaccinated as shown in the diagram. Total numbers of TgTR1 cells were evaluated in spleens on day 12. B, WT mice received Trp1-BiVax prime and 5 days later received either a BiVax/IL2Cx25 or a TriVax booster and on day 12 the numbers of Trp1 tetramer+ T cells were enumerated in spleens. C, Mean fluorescence intensity (MFI) of PD-1 expression on TgTR1 cells in the experiment in panel A. (n = 3 mice/group). D, WT mice were inoculated s.c. with 3 × 105 B16F10 cells and 7 days later mice received Trp1-BiVax vaccine prime followed by boosters with either TriVax, BiVax/IL2Cx25 or TriVax/IL2Cx25. Tumor size growth curves showing means with SD for each group (n = 5 mice/group). E, WT mice were inoculated s.c. with 3 × 105 B16F10 cells and 7 days later mice received Trp1-BiVax prime followed by booster vaccines with either TriVax, TriVax/PD-L1 (200 µg/mouse) or BiVax/IL2Cx25. Tumor size growth curves showing means with SD for each group (n = 5 mice/group). Representative results are shown from at least 3 independent experiments.

Article Snippet: Poly-ICLC was provided by Dr. Andres Salazar (Oncovir). mAb against mouse CD40 (FGK4.5, cat. # BE0016-2), mouse PD-L1 (10F.9G2, cat. # BE0101), mouse IL2 (JES6-5H4, cat. # BE0042 JES6-1A12, cat. # BE0043), mouse CD4 (GK1.5, cat. # BE0003-1), mouse CD122 (TM-beta 1, cat # BE0298), mouse CD25 (PC-61.5.3, cat # BE00012) and mouse Thy1.1 (19E12, cat. # BE0214) were purchased from BioXcell.

Techniques: Fluorescence, Expressing

Journal: Cancer immunology research

Article Title: Sustained persistence of IL2 signaling enhances the antitumor effect of peptide vaccines through T-cell expansion and preventing PD-1 inhibition

doi: 10.1158/2326-6066.CIR-17-0549

Figure Lengend Snippet: CD8+ T cells generated with BiVax/IL2Cx show resistance to PD-L1 inhibition. A, CD45.1 WT mice were vaccinated as described in Fig. 4A and on day 12 purified TgTR1 CD8+ T cells from spleens were incubated with either B16F10 (PD-L1 low) cells, IFNγ-treated B16F10 (PD-L1 high) cells (pulsed with 1 µg Trp1) with or without PD-L1 mAb (10 µg/ml) and the numbers of IFNγ producing cells were evaluated by EliSpot. B, Representative EliSpot wells from panel A. C, Diagram illustrating the artificial APCs (aAPCs) used in panel D. D, CFSE labeled previously activated TgTr1 CD8+ T cells were incubated for 72 h at a 1:1 ratio with aAPCs containing either Trp1/H2Db monomers or Trp1/H2Db monomers plus PD-L1 protein, in the presence and absence of PD-L1 mAb (10 µg/ml). Percentages of proliferating (CFSE-diluted) TgTR1 cells were evaluated by flow cytometry. E, CD45.1 WT mice were vaccinated as described in Fig. 4A and 1 h after the last dose of IL2Cx, pSTAT5 expression was evaluated in TgTR1 cells in the spleen. F, TgTR1 cells transduced or not with CA-STAT5 were adoptively transferred into WT mice, followed by a BiVax prime/boost vaccination (5 days apart). Mice receiving non-transduced T cells and treated with IL2Cx were included for comparison. On day 12, CD8+ T cells were purified and the effects of PD-L1 inhibition were evaluated as described in panel A. Representative results are shown from at least 3 independent experiments.

Article Snippet: Poly-ICLC was provided by Dr. Andres Salazar (Oncovir). mAb against mouse CD40 (FGK4.5, cat. # BE0016-2), mouse PD-L1 (10F.9G2, cat. # BE0101), mouse IL2 (JES6-5H4, cat. # BE0042 JES6-1A12, cat. # BE0043), mouse CD4 (GK1.5, cat. # BE0003-1), mouse CD122 (TM-beta 1, cat # BE0298), mouse CD25 (PC-61.5.3, cat # BE00012) and mouse Thy1.1 (19E12, cat. # BE0214) were purchased from BioXcell.

Techniques: Generated, Inhibition, Purification, Incubation, Enzyme-linked Immunospot, Labeling, Flow Cytometry, Expressing

Journal: Cancer immunology research

Article Title: Sustained persistence of IL2 signaling enhances the antitumor effect of peptide vaccines through T-cell expansion and preventing PD-1 inhibition

doi: 10.1158/2326-6066.CIR-17-0549

Figure Lengend Snippet: PEG-IL2 is effective in expanding BiVax generated antigen-specific T cells. A, WT mice (3/group) were vaccinated with BiVax prime. Five days later the mice received boosters with BiVax, BiVax/IL2, BiVax/IL2Cx122 or BiVax/PEG-IL2. Seven days later the numbers of Trp1 tetramer+ T cells were evaluated in spleens. B, WT mice were inoculated s.c. with B16F10 cells and 7 days later, received a BiVax prime followed 5 days later by booster vaccines with BiVax, BiVax/IL2 or BiVax/PEG-IL2. Tumor size growth curves showing means with SD for each group. (n = 10 mice/group). C, purified CD8+ T cells from WT mice receiving BiVax prime-boost (5 days apart) and treated or not with PEG-IL2 were incubated with either B16F10 (PD-L1 low) cells, IFNγ-treated (PD-L1 high) B16F10 cells (pulsed with 1 µg Trp1) with or without PD-L1 mAb (10 µg/ml) and the numbers of IFNγ-producing cells were evaluated by EliSpot. Representative results are shown from at least 3 independent experiments.

Article Snippet: Poly-ICLC was provided by Dr. Andres Salazar (Oncovir). mAb against mouse CD40 (FGK4.5, cat. # BE0016-2), mouse PD-L1 (10F.9G2, cat. # BE0101), mouse IL2 (JES6-5H4, cat. # BE0042 JES6-1A12, cat. # BE0043), mouse CD4 (GK1.5, cat. # BE0003-1), mouse CD122 (TM-beta 1, cat # BE0298), mouse CD25 (PC-61.5.3, cat # BE00012) and mouse Thy1.1 (19E12, cat. # BE0214) were purchased from BioXcell.

Techniques: Generated, Purification, Incubation, Enzyme-linked Immunospot