Activation of tolerogenic dendritic cells in the tumor draining lymph nodes by CD8+ T cells engineered to express CD40 ligand

Abstract

Tolerogenic dendritic cells in the tumor microenvironment can inhibit the generation and maintenance of robust antitumor T cell responses. In this study, we investigated the effects of local delivery of CD40L by tumor-reactive CD8(+) T cells on dendritic cell activation and antitumor T cell responses in the TRAMP model. To increase the immunostimulatory signal, CD40L was engineered, by deleting the majority of the cytoplasmic domain, to increase its levels of expression and duration on the surface of CD8(+) T cells. Tumor-reactive CD8(+) T cells expressing the truncated form of CD40L stimulated maturation of dendritic cells in vitro and in the prostate draining lymph nodes in vivo. Following dendritic cell maturation, a significantly higher fraction of adoptively transferred, tumor-reactive (reporter) CD8(+) T cells was stimulated to express IFN-gamma and infiltrate the prostate tissue. The antitumor CD8(+) T cell response was further enhanced if TRAMP mice were also immunized with a tumor-specific Ag. These findings demonstrate that augmented T cell responses can be achieved by engineering tumor-reactive T cells to deliver stimulatory signals to dendritic cells in the tumor microenvironment.

Figure 1. Effect of systemic anti-CD40 treatment on a tumor-reactive CD8⁺ T cell response in TRP-SY mice

TRP-SIY mice were injected with PBS or anti-CD40 antibody on days -1, 0 and +1. On day 0, mice were injected with naïve 2C cells and infected with WSN-SIY virus. Five (A) and ten (B) days post infection, single cell suspensions were prepared from the PDLN, spleens and PLN, and cells were restimulated in vitro with SIY peptide for 4hr. Samples were stained for 2C TCR, CD8 and intracellular IFN-γ. Dot plots show 2C TCR versus CD8 staining profiles of live cells. Histograms show IFN-γ expression of 2C TCR⁺CD8⁺ cells. Numbers indicate percentage of positive cells. Representative data from one of two similar experiments are shown.

Figure 2. Surface expression of wildtype and mutant CD40L

Naïve 2C cells were activated with SIY peptide plus IL-2 for 36hrs. Cells were then transduced with a retrovirus expressing Thy1.1 alone (control), Thy1.1 plus the wild type CD40L, CD40L Y5A or CD40L Δ1-13, and were analyzed for CD40L expression 24hrs and 48hrs later. The time in parentheses corresponds to days post stimulation. Dot plots show CD40L versus Thy1.1 staining profiles of live cells. Numbers indicate the percentage of Thy1.1⁺ cells that are CD40L⁺. Transduction efficiencies were 20-25% in all cases, and representative data from one of at least two similar experiments are shown.

Figure 3. Kinetics of CD40L Δ1-13 re-expression on retrovirally-transduced in vitro memory 2C cells

Naive 2C cells were activated with SIY peptide plus IL-2 for 36hrs, and were then transduced with a retrovirus expressing Thy1.1 and CD40L Δ1-13. Cells were cultured for 2 additional days in IL-2 and then transitioned into a memory phenotype by culturing them for 6 days in IL-7. In vitro memory cells were restimulated with SIY peptide plus IL-2 in the presence of a 1:1 mix of C57BL/6 splenocytes, and were stained for Thy1.1, 2C TCR, and CD40L on days 2, 3 and 4. Histograms show expression on 2C TCR⁺Thy1.1⁺ transduced cells or 2C TCR⁺Thy1.1⁻ non-transduced cells. Representative data from one of at least two similar experiments are shown.

Figure 4. CD40L Δ1-13-expressing 2C cells simulate maturation of dendritic cells in vitro and in vivo

Schematic diagram (A) and results (B) of in vitro BMDC maturation assay. Retrovirally-transduced in vitro memory 2C cells were generated as in Figure 3 and restimulated with SIY peptide for 48hrs. Thy1.1⁺ transduced 2C cells were purified by cell sorting and cultured with BMDCs for 24 hrs. Cells were stained for CD11c, CD80 and CD86. Dot plots show CD80 and CD86 expression of CD11c⁺ BMDCs under the indicated conditions. Numbers in graphs indicate the percentage of CD80⁺CD86⁺ cells. Numbers below graphs indicate the average percentage of CD80⁺CD86⁺ cells ± standard deviation (SD). Representative data from one of at least two similar experiments are shown. P value is <0.05 when percentages of CD80⁺CD86⁺ BMDCs were compared between samples stimulated with either control 2C cells or CD40L Δ1-13 2C cells. C, In vivo dendritic cell maturation. In vitro memory 2C cells either expressing or not expressing CD40L were transferred into TRP-SIY mice. Six days later, cells from the PDLN and spleens were pooled from 5 mice and stained for CD11c, CD11b, CD80 and CD86. Dot plots show CD80 and CD86 expression of CD11c⁺CD11b⁺ cells. Numbers indicate the percentage of positive cells. Representative data from one of at least two similar experiments are shown.

Figure 5. CD40L Δ1-13-expressing 2C cells stimulate a more functional anti-tumor T cell response in TRP-SIY mice

A, Schematic diagram of experimental protocol. In vitro memory 2C cells either expressing or not expressing CD40L Δ1-13 were transferred into TRP-SIY mice. As a control, some TRP-SIY mice were not injected with any in vitro memory 2C cells. The mice were divided into two groups. One group was infected intranasally with WSN-SIY and the other group was not infected. Six days later, naïve Thy1.1^High reporter 2C cells were transferred into all mice. Five days after transfer, reporter 2C cells were analyzed for IFNγ expression. B, Reporter 2C T cell responses. Cells from the PDLN and spleens of above treated mice were restimulated with SIY peptide for 4hr, and then stained for Thy1.1, CD8 and intracellular IFN-γ. Dot plots show IFN-γ versus Thy1.1 staining profiles gating on Thy1.1^High CD8⁺ reporter 2C cells. Numbers in graphs indicate the percentage of IFN-γ⁺ cells. Numbers below graphs indicate the average percentage of IFN-γ⁺ cells ± SD of the analyzed tissues. Representative data from one of two experiments are shown.