Expression of LIGHT/TNFSF14 combined with vaccination against human papillomavirus Type 16 E7 induces significant tumor regression

Abstract

LIGHT, a ligand for the lymphotoxin-beta receptor, establishes lymphoid-like tissues inside tumor sites and recruits naïve T cells into the tumor. However, whether these infiltrating T cells are specific for tumor antigens is not known. We hypothesized that therapy with LIGHT can expand functional tumor-specific CD8(+) T cells that can be boosted using HPV16E6E7-Venezuelan equine encephalitis virus replicon particles (HPV16-VRP) and that this combined therapy can eradicate human papillomavirus 16 (HPV16)-induced tumors. Our data show that forced expression of LIGHT in tumors results in an increase in expression of IFNgamma and chemoattractant cytokines such as interleukin-1a, MIG, and macrophage inflammatory protein-2 within the tumor and that this tumor microenvironment correlates with an increase in frequency of tumor-infiltrating CD8(+) T cells. Forced expression of LIGHT also results in the expansion of functional T cells that recognize multiple tumor antigens, including HPV16 E7, and these T cells prevent the outgrowth of tumors on secondary challenge. Subsequent boosting of E7-specific T cells by vaccination with HPV16-VRP significantly increases their frequency in both the periphery and the tumor and leads to the eradication of large well-established tumors, for which either treatment alone is not successful. These data establish the safety of Ad-LIGHT as a therapeutic intervention in preclinical studies and suggest that patients with HPV16(+) tumors may benefit from combined immunotherapy with LIGHT and antigen-specific vaccination.

Figure 1. Therapy of well-established C3.43 tumors with HPV16-VRP alone results in an expansion of HPV16 E7-specific T-cells but does not induce significant tumor regression

Tumor-bearing mice were treated with either VRP-CTRL (GFP-VRP) or HPV16-VRP. (A & B) Average tumor volumes ± SEM and survival are plotted against days after tumor challenge (n=10 per group). (C) Lymphocytes isolated from individual spleens (n=5 per group) or tumors of HPV16-VRP vaccinated mice were analyzed by FACS and the average frequency of CD8+/CD3+/E7-tetramer+ cells is shown. (D) IFN-gamma ELISPOT assay was used to detect functional tumor-specific T-cells after stimulation with the HPV16 E7(49-57) peptide (n=5 per group).

Figure 2. Forced expression of LIGHT induces the expansion of HPV16 E7-specific T-cells and significantly increases T-cell infiltration into C3.43 tumors

Individual spleens and draining lymph nodes were harvested 7 days after last treatment from tumor-bearing mice treated with either Ad-LIGHT or Ad-CTRL (n=5 per group). CD8+ T-cells were isolated by MACS sorting and incubated with HPV16 E7(49–57)/H-2Db tetramer, anti-CD3 and anti-CD8 antibodies. Cells were analyzed by FACS and gating was done on CD8+ cells. (A) Upper right-hand quadrant shows frequency of CD8+/CD3+/E7-tetramer+ cells in pooled lymphocytes. (B) Average frequency of CD8+/CD3+/E7-tetramer+ cells in splenocytes. (C) TILS were isolated from tumors, stained with anti-CD3 and anti-CD8 antibodies. Average frequency of CD3+CD8+ cells is shown.

Figure 3. Forced expression of LIGHT in C3.43 tumors induces increased expression of IFNg and chemokines

Three groups of 5 tumor-bearing mice were left either untreated or treated with Ad-CTRL or treated with Ad-LIGHT. On day 24, tumors were resected and homogenized. Intra-tumoral cytokines and chemokines were measured. Cytokine/chemokine concentration per gram of tumor ± SD is shown.

Figure 4. Forced expression of LIGHT in C3.43 tumors induces functional T-cells that are specific for multiple tumor antigens and increases survival of tumor-bearing mice

Groups of 5 tumor-bearing mice treated with either Ad-LIGHT or Ad-CTRL. IFN-gamma ELISPOT assay was used to detect functional peptide-specific T-cells in individual spleens. (A) HPV16 E7(49-57) (B) Ampitope (SSPVNSLRNVV) peptide (C & D) Tumor growth was monitored in groups of 10 tumor-bearing mice treated with either Ad-LIGHT or Ad-CTRL. Average tumor volumes ± SEM and survival are plotted against days after tumor challenge.

Figure 5. Forced expression of LIGHT in primary tumors prevents outgrowth of tumors upon secondary challenge

Groups of 10 tumor-bearing mice were treated with either Ad-LIGHT or Ad-CTRL. On day 24 after challenge, primary tumors were resected from the right flank and mice were given a secondary tumor challenge on the left flank. Percent survival is plotted against days after secondary tumor challenge (n=8 analyzed per group).

Figure 6. Vaccination with HPV16-VRP boosts the frequency of tumor-specific T-cells induced by intra-tumoral expression of LIGHT and leads to significant survival

Tumor-bearing mice treated with either Ad-CTRL (Ad-LacZ) or Ad-LIGHT followed by treatment with either VRP-CTRL (GFP-VRP) or HPV16-VRP. (A) TILS were analyzed by FACS and the average frequency of CD8+/CD3+/E7-tetramer+ TILS is shown (n=5 per group). (B) IFN-gamma ELISPOT assay was used to detect functional tumor-specific T-cells from individual spleens after stimulation with both the Ampitope and E7(49-57) peptides (n=5 per group). (C & D) Average tumor volumes ± SEM and survival are plotted against days after tumor challenge (n=10 per group).