Combination CTLA-4 blockade and 4-1BB activation enhances tumor rejection by increasing T-cell infiltration, proliferation, and cytokine production - PubMed (original) (raw)

Combination CTLA-4 blockade and 4-1BB activation enhances tumor rejection by increasing T-cell infiltration, proliferation, and cytokine production

Michael A Curran et al. PLoS One. 2011.

Abstract

Background: The co-inhibitory receptor Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) attenuates immune responses and prevent autoimmunity, however, tumors exploit this pathway to evade the host T-cell response. The T-cell co-stimulatory receptor 4-1BB is transiently upregulated on T-cells following activation and increases their proliferation and inflammatory cytokine production when engaged. Antibodies which block CTLA-4 or which activate 4-1BB can promote the rejection of some murine tumors, but fail to cure poorly immunogenic tumors like B16 melanoma as single agents.

Methodology/principal findings: We find that combining αCTLA-4 and α4-1BB antibodies in the context of a Flt3-ligand, but not a GM-CSF, based B16 melanoma vaccine promoted synergistic levels of tumor rejection. 4-1BB activation elicited strong infiltration of CD8+ T-cells into the tumor and drove the proliferation of these cells, while CTLA-4 blockade did the same for CD4+ effector T-cells. Anti-4-1BB also depressed regulatory T-cell infiltration of tumors. 4-1BB activation strongly stimulated inflammatory cytokine production in the vaccine and tumor draining lymph nodes and in the tumor itself. The addition of CTLA-4 blockade further increased IFN-γ production from CD4+ effector T-cells in the vaccine draining node and the tumor. Anti 4-1BB treatment, with or without CTLA-4 blockade, induced approximately 75% of CD8+ and 45% of CD4+ effector T-cells in the tumor to express the killer cell lectin-like receptor G1 (KLRG1). Tumors treated with combination antibody therapy showed 1.7-fold greater infiltration by these KLRG1+CD4+ effector T-cells than did those treated with α4-1BB alone.

Conclusions/significance: This study shows that combining T-cell co-inhibitory blockade with αCTLA-4 and active co-stimulation with α4-1BB promotes rejection of B16 melanoma in the context of a suitable vaccine. In addition, we identify KLRG1 as a useful marker for monitoring the anti-tumor immune response elicited by this therapy. These findings should aid in the design of future trials for the immunotherapy of melanoma.

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Conflict of interest statement

Competing Interests: Dr. James P. Allison is a paid consultant for Bristol-Myers Squib and is the primary inventor on the patent “Blockade of T lymphocyte down-regulation associated with CTLA-4 signaling” (US Patent 7229628; Submitted 12/07/199; Published: 06/12/2007) which has been been licensed by UC Berkeley to Bristol-Myers Squib. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1

Figure 1. Anti-CTLA-4 and α4-1BB synergize in the context of FVAX.

Kaplan-Meier survival curves for mice challenged with 2.5×104 B16-BL6 cells and vaccinated on days 3, 6 and 9 with 1×106 of A) FVAX or B) GVAX intra-dermally and the indicated antibody or combination intra-peritoneally. Lack of survival was defined as death or tumor size >1000 mm3. Each curve represents 3 independent experiments of 10 mice per group. P values were calculated using the Log-rank (Mantel-Cox) test (* - p≤0.05, ** - p≤0.01, ***-p<0.001).

Figure 2

Figure 2. Combination αCTLA-4/α4-1BB therapy promotes high effector to regulatory T-cell ratios in the tumor.

Mice were challenged with 1.5×105 B16-BL6, treated with FVAX and the indicated antibody on days 3,6 and 9, and sacrificed on day 15. Percent of CD45+ tumor infiltrating lymphocytes of A) CD8+ T-cells, B) CD4+ effector (Teff) and C) regulatory (Treg) cells is shown. The ratios of D) CD8+ T-cells to Tregs and E) CD4+ Teff to Treg are shown. Values shown are for individually analyzed mice and are the sum of 5 independent experiments with 5–15 mice per group. The ratios of F) CD8+ T-cells to Tregs and G) CD4+ Teff to Treg are shown at Day 25 for individual mice from 2 independent experiments. Student's t-tests were performed to determine statistical significance between samples (* - p≤0.05, ** - p≤0.01, ***-p<0.001).

Figure 3

Figure 3. 4-1BB activation cooperates with CTLA-4 blockade to induce peripheral inflammatory cytokine production.

Mice challenged with 2.5×105 B16-OVA cells and treated on days 6, 9 and 12, were sacrificed on Day 14. T-cells were purified from tumor-draining (TDLN) and vaccine-draining (VDLN) lymph nodes, stained with antibodies, and sorted by flow cytometry into CD4+ and CD8+ subsets. Cytokine production was measured after 36 hours using the TH1/TH2/TH17 CBA Kit (BD) and is shown for A) 2×105 VDLN and B) 2×105 TDLN CD8 T-cells restimulated on 1×105 OVA 257–264 peptide pulsed DCs and for C) 2.5×105 CD4 T-cells restimulated on 1×105 OVA 323–339 peptide pulsed DCs. Data is shown for 3–4 independent experiments with 5–10 pooled mice per group.

Figure 4

Figure 4. Combination αCTLA-4/α4-1BB therapy enhances intra-tumoral T-cell cytokine production and proliferation.

Mice challenged with 2.5×105 B16-OVA cells and treated on days 6, 9 and 12, were sacrificed on Day 14. TIL were purified from 5–10 pooled tumors per group and enriched using the Miltenyi T-cell purification kit. 2×106 TIL were restimulated with 7.5×105 DC (1∶1 mix of MHC-I and MHC-II peptide pulsed DCs) for 8 hours in the presence of BD GolgiPlug. Cells were fixed using the FoxP3 kit and analyzed by flow cytometry for lymphocyte markers and intracellular IFN-γ and TNF-α production for A) CD8s and B) CD4 Teffs and also Ki67 expression for C) CD8s and D) CD4 Teffs. Data shown is from 4 independent experiments. All means shown are +/− S.E.M.

Figure 5

Figure 5. Combination αCTLA-4/α4-1BB therapy induces KLRG1 and PD-1 expression by tumor-infiltrating T-cells.

Mice challenged with 1.5×105 B16-BL6 cells and treated on days 3, 6 and 9, were sacrificed on Day 15. TIL were fixed and stained for lymphocyte lineage and activation markers using the FoxP3 fixation kit. Percent of A) CD8+ T-cells, B) CD4+ effector (Teff) and C) regulatory (Treg) cells expressing KLRG1, PD-1, and CTLA-4 are shown. The 4F10 clone of αCTLA-4 was used to detect cells on which CTLA-4 had been blocked in vivo using the 9D9 clone. Values shown are for individually analyzed mice and are the sum of 4–6 independent experiments with 5–15 mice per group. Student's t-tests were performed to determine statistical significance between samples(* - p≤0.05, ** - p≤0.01, ***-p<0.001).

Figure 6

Figure 6. High KLRG1 expression by tumor-infiltrating effector T-cells at Day-25 in mice treated with combination αCTLA-4/α4-1BB therapy.

Mice challenged with either 2.5×104 (Untreated and FVAX), 1.0×105 (FVAX+αCTLA-4 or α4-1BB), or 2.0×105 (FVAX+αCTLA-4/α4-1BB) B16-BL6 cells and treated on days 4, 7 and 10, and 13 were sacrificed on Day 25. TIL were fixed and stained for lymphocyte lineage and activation markers using the FoxP3 fixation kit. Percent of A) CD8+ T-cells, B) CD4+ effector (Teff) expressing KLRG1 are shown. Values shown are for individually analyzed mice and are the sum of 2 independent experiments with 10 mice per group. Student's t-tests were performed to determine statistical significance between samples(* - p≤0.05, ** - p≤0.01, ***-p<0.001).

Figure 7

Figure 7. Combination αCTLA-4/α4-1BB therapy induces higher tumor infiltration by CD4+KLRG1+ T-cells than α4-1BB alone.

Mice challenged with 1.5×105 B16-BL6 cells and treated on days 3, 6 and 9, were sacrificed on Day 15. The number of KLRG1+ T-cells of a given lineage was calculated by multiplying the %KLRG1+ TIL determined by flow cytometry by the measured number of CD45+ lymphocytes per mm3 of tumor. Data shown are ratios of the absolute number of KLRG1+ T-cells from combination treated tumors to the number of KLRG1+ T-cells from α4-1BB alone treated tumors. Ratios were calculated for 5 independent experiments with 5–15 mice per group.

Figure 8

Figure 8. Combination αCTLA-4/α4-1BB treatment generates more pro-inflammatory ratios of effector T-cells to MDSC in the context of FVAX versus GVAX.

Mice challenged with 1.5×105 B16-BL6 cells and treated on days 3, 6 and 9, were sacrificed on Day 15. TIL were fixed and stained for myeloid and lymphocyte lineage and activation markers using the FoxP3 fixation kit. The ratios of A) CD8+ T-cells to CD11b+GR-1+ MDSC and B) CD4+ Teff to MDSC are shown in the FVAX setting. Values shown are for individually analyzed mice and are the sum of 3 independent experiments with 5–15 mice per group. The ratios of C) CD8+ T-cells to MDSC and D) CD4+ effector T-cells to MDSC are shown in the context of GVAX for 10 individual mice per group. Student's t-tests were performed to determine statistical significance between samples (* - p≤0.05, ** - p≤0.01, ***-p<0.001).

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