Adjuvant IL-15 does not enhance the efficacy of tumor cell lysate-pulsed dendritic cell vaccines for active immunotherapy of T cell lymphoma

Abstract

There has been a recent interest in using IL-15 to enhance antitumor activity in several models because of its ability to stimulate CD8+ T cell expansion, inhibit apoptosis and promote memory T cell survival and maintenance. Previously, we reported that C6VL tumor lysate-pulsed dendritic cell vaccines significantly enhanced the survival of tumor-bearing mice by stimulating a potent tumor-specific CD8+ T cell response. In this study, we determined whether IL-15 used as immunologic adjuvant would augment vaccine-primed CD8+ T cell immunity against C6VL and further improve the survival of tumor-bearing mice. We report that IL-15 given after C6VL lysate-pulsed dendritic cell vaccines stimulated local and systemic expansion of NK, NKT and CD8+ CD44hi T cells. IL-15 did not, however, augment innate or cellular responses against the tumor. T cells from mice infused with IL-15 following vaccination did not secrete increased levels of tumor-specific TNF-alpha or IFN-gamma or have enhanced C6VL-specific CTL activity compared to T cells from recipients of the vaccine alone. Lastly, IL-15 did not enhance the survival of tumor-bearing vaccinated mice. Thus, while activated- and memory-phenotype CD8+ T cells were dramatically expanded by IL-15 infusion, vaccine-primed CD8+ T cell specific for C6VL were not significantly expanded. This is the first account of using IL-15 as an adjuvant in a therapeutic model of active immunotherapy where there was not a preexisting pool of tumor-specific CD8+ T cells. Our results contrast the recent studies where IL-15 was successfully used to augment tumor-reactivity of adoptively transferred transgenic CD8+ T cells. This suggests that the adjuvant potential of IL-15 may be greatest in settings where it can augment the number and activity of preexisting tumor-specific CD8+ T cells.

Fig. 1

Interleukin-15 infusion results in increased cell numbers in the DLN, CLLN and spleen. Mice bearing a lethal number of C6VL cells (i.p.) were vaccinated twice s.c. with 5.0×10⁵ C6VL-DC or HBSS (control). 0.5 μg rhIL-15 was administered s.c. at the vaccine site every 12 h for 5 days following each vaccination. Mice not receiving IL-15 were infused with only the diluent. Eighteen hours following the last infusion, DLN, CLLN and spleens were harvested from mice and viable cells enumerated. Bars represent the average number of cells (×10⁶) isolated per DLN, CLLN or spleen (n=3–10 mice/group) in 2 (CLLN) or 4 (DLN and spleen) independent experiments ± SD. P-values were calculated using a 2-sample Student’s t-test (*P<0.012; **P=0.015, ^#,## P<0.05)

Fig. 2

Recipients of s.c. IL-15 have increased numbers of NK, NKT and T cells in their DLN. Tumor-bearing mice were vaccinated twice s.c. with 5.0×10⁵ C6VL-DC or HBSS only. Mice were infused s.c. with IL-15 or diluent only every 12 h for 5 days after each vaccine. Eighteen hours following the last infusion, DLN were harvested and analyzed by flow cytometry for cell surface expression of markers indicative of (b) NK, NKT, and αβ T cells; or (a) and (c) CD4⁺ and CD8⁺ T cells subsets as described in Materials and methods. Average absolute numbers of each cell type per DLN were calculated as [(percentage of total cells staining positive × number of viable cells isolated)/ n of mice per group]. a Representative primary flow cytometry data monitoring the percents of CD4 and CD8 T cells in the DLN of C6VL-DC recipients +/- IL-15. b and c Bars represent the average absolute numbers of cell types in the DLN of each group (n=5–10 mice/group) in four independent experiments ± SD. d Bars represent the ratio of CD4⁺ to CD8⁺ T cells in the DLN of each group

Fig. 3

Antigen-activated or memory-phenotype CD8⁺ T cells are expanded by IL-15 infusion. Groups of 10 (+IL-15) or 20 (no IL-15) tumor-bearing mice were vaccinated and infused with IL-15 as indicated in the Materials and methods. Eighteen hours following the last IL-15 injection, DLN were harvested and CD8⁺ T cells purified by negative selection. The CD8⁺ T cells were stained for cell surface expression of CD8b (PerCP-Cy5.5) and CD44 (FITC) alone (b), or in combination with PE-conjugated CD62L, CD122, or CCR7 (a–c) and analyzed by flow cytometry. Bars indicate (b) the average (%) (left) and absolute number (right) of CD8b⁺ CD44^hi cells per DLN in each treatment group, or c, d the percentage of CD8b⁺ cells in the DLN that are both CD44hi and CD62L^+/−, CD122⁺ or CCR7⁺ . P values were calculated using a two sample Student’s t test (* and **P<0.0001)

Fig. 4

Interleukin-15 infusion does not increase vaccine-primed, tumor-specific cytokine release by DLN cells. Groups of five mice were vaccinated with C6VL-DC or HBSS and infused with rhIL-15 as described in Materials and methods. Eighteen hours following the last IL-15 infusion, DLN were harvested and stimulated for 72 h in vitro with mitomycin-C-treated C6VL or control tumor cells at a ratio of one tumor cell per five responder cells. Cytokine concentrations in culture supernatants were measured as described in the Materials and methods. Bars represent the mean of triplicate samples ±SD. The dotted line represents the assays lower limit of detection (20 pg/ml). P values were calculated using a two sample Student’s t test. Cytokine secretion by mitomycin-C-treated tumor cells and DLN from recipients of only IL-15 or its diluent were below the limits of detection in all cases. Representative data from one of three independent experiments is shown

Fig. 5

Interleukin-15 infusions do not stimulate NK-mediated killing of C6VL. Six tumor-bearing mice per group were vaccinated with C6VL-DC or HBSS and infused with IL-15 as indicated in the Materials and methods. Mice were infused s.c. with 0.5 μg rhIL-15 every 12 h for five consecutive days following each vaccine. Eighteen hours following the last IL-15 infusion, DLN and spleens were harvested, pooled and used immediately in an overnight CTL assay. Specific lysis of DiO-labeled C6VL (a) and NK-susceptible YAC-1 (b) tumor cells at E:T of 80:1, 20:1 and 5:1 were assessed by flow cytometry following a 16-h incubation of targets with effectors. Percent lysis was calculated as described in the Materials and methods. Data points represent the mean of duplicate samples ±SD. Representative data from one of three independent experiments is shown

Fig. 6

Interleukin-15 does not augment the tumor-specific CTL activity stimulated by C6VL-DC vaccines in tumor-bearing mice. Six mice per group were vaccinated with C6VL-DC or HBSS and were given IL-15 or diluent only as detailed in the Materials and methods. Eighteen hours following the last IL-15 infusion, DLN, splenocytes and peritoneal lavage samples were harvested and restimulated in vitro for 5 days with one irradiated C6VL cell per five responder cells. Specific lysis of DiO-labeled C6VL and MBL-2 (control) tumor cells by (a), DLN; (b), splenocytes; or c, peritoneal lavage cells at E:T of 50:1, 17:1 and 5.6:1 was assessed by flow cytometry following a 4-h incubation of targets with restimulated effectors. Percent lysis was calculated as described in the Materials and methods. Data points represent the mean of duplicate samples ±SD. P values were calculated using a two sample Student’s t test at each data point. Representative data from one of two independent experiments is shown

Fig. 7

Interleukin-15 infusion does not enhance the survival of tumor-bearing C6VL lysate-pulsed DC vaccine recipients. Naïve mice were challenged with a lethal dose of C6VL. Twenty-four hours following tumor challenge, mice received their first vaccine of 5.0×10⁵ C6VL or MBL-2 lysate-pulsed DC. The vaccines continued every 10 days for three vaccines. Beginning 1d following each vaccine and continuing every 12 h for 5 days, 0.5 μg rhIL-15 was administered s.c. at the vaccine site. Control mice receiving DC vaccines but not IL-15 were injected with diluent alone on the same schedule. The mice were monitored for survival for 60 days following tumor challenge

Fig. 8

C6VL-DC+IL-15 recipients do not have enhanced C6VL-specific CTL activity per-DLN or per-CD8⁺ T cell compared to recipients of C6VL-DC vaccines alone. Tumor-bearing donor mice (n=13 per group) were vaccinated twice with 5.0×10⁵ C6VL- or MBL-2-lysate-pulsed DC and infused s.c. with 0.5 μg rhIL-15 twice daily for 5 days following each vaccine. Mice vaccinated with C6VL-DC or MBL-2-DC without IL-15 infusion (n=11 per group) were control donors. Eighteen hours following the last IL-15 infusion, DLN from each group were harvested, pooled, mixed with C6VL cells and injected into the peritoneum of ten naïve recipient mice per group. Each naïve recipient received 5000 C6VL cells and either a a number of cells equivalent to one donor’s DLN (3.2×10⁶, C6VL-DC and MBL-2-DC DLN; or 6.7×10⁶, C6VL-DC+IL-15 and MBL-2-DC+IL-15 DLN) or b a number of cells from IL-15 recipient DLN that resulted in the transfer of a number of CD8⁺ T cells that matched those transferred in 1 DLN from C6VL-DC and MBL-2-DC donors (1.7×10⁶ total cells or 5.9×10⁵ CD8⁺ per recipient)