Journal: Blood

Article Title: Accumulation of 4-1BBL + B cells in the elderly induces the generation of granzyme-B + CD8 + T cells with potential antitumor activity

doi: 10.1182/blood-2014-03-563940

Figure Lengend Snippet: 4BL cells induce GrB+CD8+ T cells. (A) Old human B cells induce higher GrB expression in target CD8+ T cells from young donors in mixed lymphocyte reaction (MLR). Shown GrB and IFNγ induction in CD8+ T cells (i, representative dot plot, %) and its mean (ii, % after deducting background response without B cells, No B) ± SEM examined in triplicate experiments and reproduced at least 3 times. (B) Positive correlation (P < .0001) between the induction of GrB (y-axis) in young CD8+ T cells and 4-1BBL expression levels on B cells (x-axis). (C) Murine 4BL cells induce GrB in TCR transgenic pmel CD8+ T cells by presenting cognate antigen (gp10025-32 peptide). eFluor670-labeled pmel CD8+ T cells were in vitro stimulated with B cells from young, Old-IgG, and Old-restored mice (as in Figure 2D) pulsed with gp10025-32 (gray bars) or control SPANX peptide (open bars). Shown is the mean ± SEM (%) of GrB within proliferating pmel CD8+ T cells in triplicate experiments reproduced 3 times. (D) ABCs (sort-purified, open bars) from young and old mice (as in Figure 2C) were compared with total B cells (gray bars) for the ability to induce GrB+CD8+ T cells stimulated with anti-CD3 Ab. (E) To demonstrate the 4BL cell-induced in vivo expansion of GrB+CD8+ T cells, naïve old mouse B cells and eFluor670-labeled CD8+ T cells from naïve congenic mice were i.v. injected into μMT mice bearing B16 melanoma (schema). One week later, GrB was quantified within proliferating LN CD8+ T cells using gp10025-32 dextramer. Shown are a representative dot plot (i-ii, %) and mean ± SEM (ii, %) of a 4 to 5-mice-per-group experiment independently reproduced 2 times.

Article Snippet: After 7 days, CD8 + T cells were quantified using gp100 dextramer IMDQVPFSV (Immudex, Copenhagen, Denmark) or Vβ13-PE Ab (clone MR12-4, BioLegend).

Techniques: Expressing, Transgenic Assay, Labeling, In Vitro, Control, Purification, In Vivo, Injection

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Peptide-loaded Langerhans cells, despite increased IL15 secretion and T cell activation in vitro , elicit anti-tumor T cell responses comparable to peptide-loaded monocyte-derived dendritic cells in vivo

doi: 10.1158/1078-0432.CCR-10-3421

Figure Lengend Snippet: Responses against peptide-loaded moDCs or LCs were measured at baseline prevaccine and again approximately three weeks after each of three vaccines given at nearly four week intervals. Responses were based on a single 6-7 day restimulation in vitro, using the same moDCs or LCs used to vaccinate a given patient, pulsed with the indicated peptides. No exogenous cytokines were added. Box plots show the medians and interquartile ranges (25th to 75th percentiles) with whiskers approximating +/− 2 SD or 95% of the data (Tukey method). (A) Absolute numbers of CD3+ CD8+ T cells reactive with tyrosinase-, gp100-, or fluMP-HLA-A*0201 tetramers are shown over time, where positive events exhibited at least one log higher fluorescent intensity than the negative controls. (B) The fold increases in IFN-gamma secretion over time by total T cells in ELISpot assays, relative to the prevaccine baseline average + 2 SDs, are depicted. Empty moDC targets without peptides were associated with high background IFN-gamma secretion in the ELISpot assay, so the background values were subtracted from all conditions at all timepoints to calculate fold increases. LCs were compared with moDCs where the outcomes were reactivity against tyrosinase, gp100, or control fluMP. (A) and (B) The test was stratified by vaccine number over time, and a permutation test generated P values. Only tyrosinase-HLA-A*0201 tetramer reactivity achieved significance in favor of LCs over moDCs (P = 0.04). There was a trend in favor of moDCs in the ELISpot assays for tyrosinase-specific responses (P = 0.08). P = NS for all other comparisons between LCs and moDCs.

Article Snippet: Antigen-loading of moDCs or LCs The clinical trial used two synthetic, HLA-A*0201-restricted, heteroclitic melanoma peptides: tyrosinase (TYR368-376) YMDGTMSQV and gp100 (gp100209-217) IMDQVPFSV (Research Genetics, Invitrogen, Carlsbad, CA).

Techniques: In Vitro, Enzyme-linked Immunospot, Generated

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Peptide-loaded Langerhans cells, despite increased IL15 secretion and T cell activation in vitro , elicit anti-tumor T cell responses comparable to peptide-loaded monocyte-derived dendritic cells in vivo

doi: 10.1158/1078-0432.CCR-10-3421

Figure Lengend Snippet: PBMCs from melanoma patients previously vaccinated with peptide-pulsed, mature, autologous, dendritic cells (LCs or moDCs) were restimulated in vitro by the same type of autologous dendritic cell pulsed with (A) fluMP or (B) gp100 peptide. Following three weekly restimulations, ELISpot assays measured IFN-gamma secretion after overnight exposure to the respective peptide-pulsed dendritic cell targets. Limited cell numbers precluded isolation of CD3+CD8+ T cells for the ELISpot assays. IL15 was supplemented at the indicated concentrations during the restimulations in vitro. Shown are the averaged triplicate means +/− SEM (n=3 independent experiments) for the number of IFN-gamma spot forming cells (SFC) per triplicate of 105 input cells. By Student’s t test, P < 0.05 LCs vs moDCs at each IL15 concentration tested. Only LCs were still stimulatory in the absence of exogenous IL15. (See also Figure 4C, which validates the concordance between IFN-gamma secretion and actual target lysis by CTLs). (C) PBMCs from melanoma patients, previously vaccinated with either peptide-pulsed, mature, autologous LCs or moDCs, were restimulated in vitro by the same type of autologous dendritic cells pulsed with gp100 peptide over each of two weeks (stimulator:responder = 1:30). No exogenous cytokines, including IL15, were added to any condition. The peptide-pulsed LCs and moDCs were opsonized at the outset and upon each restimulation with anti-IL15R-alpha or isotype control. IFN-gamma secretion by PBMCs was analyzed by ELISpot after overnight exposure to the same peptide-pulsed LC or moDC targets. Shown are the averaged triplicate means +/− SEM (n=3 independent experiments) for the number of IFN-gamma spot forming cells (SFC) per triplicate of 105 input cells. By Student’s t test, *P < 0.03 for LCs and **P < 0.0001 for moDCs, with vs without blockade of IL15R-alpha.

Article Snippet: Antigen-loading of moDCs or LCs The clinical trial used two synthetic, HLA-A*0201-restricted, heteroclitic melanoma peptides: tyrosinase (TYR368-376) YMDGTMSQV and gp100 (gp100209-217) IMDQVPFSV (Research Genetics, Invitrogen, Carlsbad, CA).

Techniques: In Vitro, Enzyme-linked Immunospot, Isolation, Concentration Assay, Lysis

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Peptide-loaded Langerhans cells, despite increased IL15 secretion and T cell activation in vitro , elicit anti-tumor T cell responses comparable to peptide-loaded monocyte-derived dendritic cells in vivo

doi: 10.1158/1078-0432.CCR-10-3421

Figure Lengend Snippet: (A) CD3+CD8+ T cells were gated from PBMCs obtained from melanoma patients previously vaccinated with tyrosinase, gp100, and flu matrix peptide-pulsed moDCs in vivo. CD3+CD8+ T cells (X axis) that reacted with HLA-A*0201 tetramers bearing the respective peptides (Y axis) were measured at the outset of each restimulation by autologous peptide-pulsed moDCs in vitro. Dot plots show only the combined IL2 (10 IU/ml) + IL15 (10 ng/ml) condition. Numbers in the right upper quadrants represent the percentages of tetramer-reactive cells in the CD3+CD8+ gate. Quadrants are the same for each round of stimulation in a single column but different across rows, because staining and analyses occurred at different time points after each round of stimulation. These data represent one of three independent experiments. (B) ELISpot assays measuring IFN-gamma secretion by responder PBMCs tested as effectors against gp100 peptide, tyrosinase peptide, and fluMP-pulsed moDC targets were performed after the first and third stimulations. Shown are the results for the gp100 target antigen, but the other two antigens stimulated similar response patterns (not shown). The averaged triplicate means ± SEM of IFN-gamma spot forming cells (SFC) per 105 input T cells combined from three independent experiments are shown. **P < 0.005 for the combination of IL2 + IL15 relative to all other conditions after the third round of stimulation. *P < 0.05 relative to the IL2 or no added exogenous cytokine conditions for IL15 either alone at both time points or in combination with IL2 after the first stimulation. (C) gp100-specific CD8+ T cells that developed in the presence of IL2 (10 IU/ml) and IL15 (10ng/ml) killed HLA-A*0201-transfected K562 targets (gift of Dr. Thomas Wolfel, Univ. Mainz, Germany (37)) pulsed with gp100 but not with tyrosinase peptide in a 51Cr release assay. The triplicate means for specific lysis {({sample release - spontaneous release}/{total release – spontaneous release}) × 100%} were averaged from three independent experiments and plotted +/− SEM, P <0.001. (D) The percentages of tyrosinase tetramer-reactive cells that also expressed each of the epitopes listed along the X-axis were pooled from three independent experiments (mean +/− SD). (E) Dot plots from one of the three experiments summarized in (D). Numbers in the upper quadrants indicate the percentages of tetramer reactive cells that did or did not express the respective epitope indicated on the X-axis. While the proportion expressing CD27 was quite variable over the three separate experiments, as shown in (D), the tetramer-reactive T cells from this experiment displayed a very clear population of CD27+ cells.

Article Snippet: Antigen-loading of moDCs or LCs The clinical trial used two synthetic, HLA-A*0201-restricted, heteroclitic melanoma peptides: tyrosinase (TYR368-376) YMDGTMSQV and gp100 (gp100209-217) IMDQVPFSV (Research Genetics, Invitrogen, Carlsbad, CA).

Techniques: In Vivo, In Vitro, Staining, Enzyme-linked Immunospot, Transfection, Release Assay, Lysis, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Peptide-loaded Langerhans cells, despite increased IL15 secretion and T cell activation in vitro , elicit anti-tumor T cell responses comparable to peptide-loaded monocyte-derived dendritic cells in vivo

doi: 10.1158/1078-0432.CCR-10-3421

Figure Lengend Snippet: (A) Responder T cells underwent a single round of stimulation by autologous gp100-pulsed moDCs and were assessed for cell death by flow cytometry after gating on the CD3+CD8+ population. The percentages of Annexin-V+ and either PIneg (early apoptotic) or PI+ (late apoptotic) were summed to obtain the total percentage of apoptotic cells. Data represent the averages of duplicate means ± SEM from 6 independent experiments. P < 0.05 for either IL15 condition with or without IL2, compared with IL2 alone or no exogenous cytokine condition. (B) Western blot analysis of Bcl-2 was performed on total cell lysates from CD3+CD8+ T cells purified by positive immunomagnetic selection and stimulated for 7 days by gp100-pulsed autologous moDCs in the presence of different cytokine conditions. CD3+CD8+ T cells originated from a previously vaccinated melanoma patient (pt) or from a healthy donor (nl). Results from one of 3 independent experiments are shown. GAPDH in CD3+CD8+ T cells served as an internal control. (C) T cells were restimulated in vitro twice over 7d each by either autologous (C) gp100- or (D) fluMP-pulsed moDCs, using the indicated cytokine conditions. Bar graphs represent the proportion of phenotypic Tregs based on CD4+CD25bright T cells that coexpressed intracellular Foxp3 (means +/− SD from 3-4 independent experiments). Insets depict the Foxp3+ expression by these gated CD4+CD25bright T cells from a representative condition without cytokine. P < 0.01 for pairwise comparisons between either of the IL15-containing and the IL2 alone or no cytokine conditions.

Article Snippet: Antigen-loading of moDCs or LCs The clinical trial used two synthetic, HLA-A*0201-restricted, heteroclitic melanoma peptides: tyrosinase (TYR368-376) YMDGTMSQV and gp100 (gp100209-217) IMDQVPFSV (Research Genetics, Invitrogen, Carlsbad, CA).

Techniques: Flow Cytometry, Western Blot, Purification, Selection, In Vitro, Expressing

Journal: Immunologic research

Article Title: Increased effector-target cell conjugate formation due to HLA restricted specific antigen recognition

doi: 10.1007/s12026-008-8041-1

Figure Lengend Snippet: Morphologic and flow cytometry characteristics of conjugates in gp100 positive melanoma cell lines without and with HLA- A*0201 allele. (a) 400×, less 1520-TIL bound to a single 624.28-MEL; (b) 400×, more 1520-TIL bound to a single 624.38-MEL; dot plot analysis of co-culture consisting of: (c) 1520-TIL and 624.28-MEL and (d) 1520-TIL and 624.38-MEL

Article Snippet: The gp100:209–217 (210M) (IMDQVPFSV, gp100–209 2M) peptide was commercially synthesized by Princeton Biomolecules (Columbus, OH).

Techniques: Flow Cytometry, Co-Culture Assay

Journal: Immunologic research

Article Title: Increased effector-target cell conjugate formation due to HLA restricted specific antigen recognition

doi: 10.1007/s12026-008-8041-1

Figure Lengend Snippet: Percentages of free and conjugate particles in gp100 positive melanoma cell lines with or without HLA-A*0201 allele

Article Snippet: The gp100:209–217 (210M) (IMDQVPFSV, gp100–209 2M) peptide was commercially synthesized by Princeton Biomolecules (Columbus, OH).

Techniques:

Journal: Immunologic research

Article Title: Increased effector-target cell conjugate formation due to HLA restricted specific antigen recognition

doi: 10.1007/s12026-008-8041-1

Figure Lengend Snippet: Percentages of free and conjugate particles in 1390-MEL pulsed with soluble gp100 peptide, control flu peptide, or no peptide

Article Snippet: The gp100:209–217 (210M) (IMDQVPFSV, gp100–209 2M) peptide was commercially synthesized by Princeton Biomolecules (Columbus, OH).

Techniques: Control

Journal: Immunologic research

Article Title: Increased effector-target cell conjugate formation due to HLA restricted specific antigen recognition

doi: 10.1007/s12026-008-8041-1

Figure Lengend Snippet: Correlation between percentage of lymphocytes in conjugates and those with increased intracellular IFN-γ production. The dark bars represent the percentages of lymphocytes in conjugates, and the shaded bars represent the percentages of lymphocytes producing intracellular IFN-γ. The representative results from the following co-cultures are summarized in (a). 1520-TIL and 624.38-MEL; (b) 1520-TIL and 624.28-MEL; (c) 1520-TIL and 1390-MEL pulsed with gp100–209 2M peptide; and (d) 1520-TIL and 1390-MEL without peptide pulsing. The background from antigen-independent adhesions has been subtracted from the values in Fig. 2a and c

Article Snippet: The gp100:209–217 (210M) (IMDQVPFSV, gp100–209 2M) peptide was commercially synthesized by Princeton Biomolecules (Columbus, OH).

Techniques:

Journal: Immunologic research

Article Title: Increased effector-target cell conjugate formation due to HLA restricted specific antigen recognition

doi: 10.1007/s12026-008-8041-1

Figure Lengend Snippet: Inhibition of conjugate formation and intracellular IFN- γ production by HLA Class I MAB

Article Snippet: The gp100:209–217 (210M) (IMDQVPFSV, gp100–209 2M) peptide was commercially synthesized by Princeton Biomolecules (Columbus, OH).

Techniques: Inhibition, Control