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

Abstract

Purpose: We compared the efficacy of human Langerhans cells (LC) as tumor immunogens in vivo with monocyte-derived dendritic cells (moDC) and investigated how interleukin 15 (IL15) supports optimal DC-stimulated antitumor immunity.

Experimental design: American Joint Committee on Cancer stage III/IV melanoma patients participated in this first clinical trial comparing melanoma peptide-pulsed LC with moDC vaccines (NCT00700167, www.ClinicalTrials.gov). Correlative studies evaluated mechanisms mediating IL15 support of DC-stimulated antitumor immunity.

Results: Both DC vaccines were safe and immunogenic for melanoma antigens. LC-based vaccines stimulated significantly greater tyrosinase-HLA-A*0201 tetramer reactivity than the moDC-based vaccines. The two DC subtypes were otherwise statistically comparable, in contrast to extensive prior data in vitro showing LC superiority. LCs synthesize much more IL15 than moDCs and stimulate significantly more antigen-specific lymphocytes with a cytolytic IFN-γ profile even without exogenous IL15. When supplemented by low-dose IL15, instead of IL2, moDCs stimulate 5 to 6 logs more tumor antigen-specific effector memory T cells (T(EMRA)) over 3 to 4 weeks in vitro. IL2 and IL15 can be synergistic in moDC stimulation of cytolytic T cells. IL15 promotes T-cell expression of the antiapoptotic bcl-2 and inhibits candidate regulatory T-cell (Treg) expansion after DC stimulation, countering two effects of IL2 that do not foster tumor immunity.

Conclusions: MoDC-based vaccines will require exogenous IL15 to achieve clinical efficacy. Alternatively, LCs can couple the endogenous production of IL15 with potent T-cell stimulatory activity. Optimization of full-length tumor antigen expression for processing into multiple immunogenic peptides for presentation by both class I and II MHC therefore merits emphasis to support more effective antitumor immunity stimulated by LCs.

Figures

Figure 1. Peptide-pulsed moDC and LC vaccines stimulate delayed type hypersensitivity reactions and KLH-specific responses after the second and third vaccines

Erythema and induration were measured at the greatest diameter around each of ten injection sites to calculate the mean for each parameter for each patient after vaccine #2 and again after vaccine #3. Shown are the averages +/− SD of the individual patient means (n = 12 patients) for each subtype DC vaccine. P = NS comparing moDCs with LCs. PBMCs from moDC and LC vaccinees were harvested at baseline prevaccine and approximately three weeks after each of three vaccines and cultured with KLH (1μg/ml). KLH-specific immunity was determined based on the incorporation of 3HTdR by proliferating responder T cells (y-axis, log2 scale) in the last 8 h of a 5-day culture.

Figure 2. Peptide-loaded moDCs and LCs generate immune responses in patients with advanced melanoma, but neither is clearly superior in this setting

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.

Figure 3. LCs are more potent than moDCs in inducing IFN-gamma-secreting CTL at limiting doses of exogenous IL15

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.

Figure 4. IL15 and IL2 synergize in supporting development of moDC-stimulated, antigen specific CD3+CD8+ T cells with an IFN-gamma-secreting, cytolytic profile and effector memory phenotype

(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.

Figure 5. IL15 helps rescue CD3+CD8+ T cells from IL2 induced apoptotic cell death and reduces the frequency of CD4+CD25bright Foxp3+ regulatory T cells after moDC stimulation

(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.