Tumor cells, rather than dendritic cells, deliver antigen to the lymph node for cross-presentation

Abstract

It is widely accepted that generation of tumor specific CD8+ T-cell responses occur via cross-priming; however the source of tumor antigen for this event is unknown. We examined the source and form of tumor antigen required for cross-presentation in the local lymph node (LN) using a syngeneic mouse tumor model expressing a marker antigen. We found that cross-presentation of this model tumor antigen in the LN is dependent on continuous traffic of antigen from the tumor site, but without any detectable migration of tumor resident dendritic cells (DCs). Instead, small numbers of tumor cells metastasize to local LNs where they are exposed to a localized CTL attack, resulting in delivery of tumor antigen into the cross-presentation pathway.

Figure 1. Cross-presentation of tumor antigen in the TDLN is dependent on DCs and persists following tumor removal. A, TDLNs were removed from mice bearing AB1HA mice on day 21 post inoculation and separated into DC-enriched and DC-depleted fractions using CD11c microbeads. DCs were co-cultured with CFSE-labeled CD8⁺ T cells and analyzed by flow cytometry after 60h. Plot depicts the percentage of gated proliferating CD8⁺CFSE⁺ CL4 T cells. Mean ± SEM, Students T test. B, AB1-HA tumors were surgically removed on day 16 post inoculation, and CFSE-labeled CL4-TCR transgenic lymphocytes adoptively transferred at the indicated time points. Percentage of gated proliferating CD8⁺CFSE⁺ CL4 T cells plotted against the day of adoptive transfer for mice. Circles represent individual mice. Students T test comparing day of surgery (D.O.S) with indicated time points post-surgery.

Figure 2. DCs do not migrate from the tumor site. Naïve and AB1-HA bearing mice were injected with 2x10⁷ fluoresbrite carboxy YG microbeads s.c. or i.t. respectively. One and three days later, tissues were harvested, stained with anti-CD11c Ab and analyzed by flow cytometry. A, Representative profiles of tumor, TDLN and normal LN DCs at days 1 and 3 post injection. Bead+ DCs gated as indicated. B, Number of tumor infiltrating bead+ DCs at days 1 and 3 post injection, Four mice per group. C, Number of bead+ DCs in the TDLN (ie. arriving from the s.c. tumor) vs. normal LN (i.e., arriving from tumor-free skin) at days 1 and 3 post injection. Four mice per group.

Figure 3. Tumor antigen is associated with DC in the TDLN. Balb/c mice were injected s.c. with DiI-labeled AB1HA tumor cells. One 3, 8 and 14 d later, axillary and inguinal TDLN were harvested. Following enzymatic digestion, lymph nodes were stained with CD11c specific mAbs and the number of DiI-positive cells determined by flow cytometry. A, Representative profiles of CD11c vs. DiI label in the axillary and inguinal TDLN. Gates show the percentage if DiI+ nonDCs (left) and DiI+ DCs (right). B and C, Number of DiI+ DCs or nonDC at days 0, 1, 3, 8 and 14 post injection in the axillary (B) and inguinal (C) TDLNs. Six mice per group. Mean ± SEM. Data shown is pooled from two individual experiments.

Figure 4. Destruction of occult tumor cells in TDLNs is dependent on T cells. A, PCR analysis of HA (upper) and GAPDH (lower) expression in TDLN of mice bearing day 21 AB1-HA tumors. Representative of 2 individual experiments. B, Limit of detection of AB1HA tumor cells in LN preparations by PCR. C, Amount of HA-specific CD8⁺ T cell killing in vivo in the TDLN, nonDLN and spleen of AB1-HA bearing mice at day 16 post inoculation. Dots represent individual mice. D, Representative pictures showing AB1-HA tumor cells grown out from cultures of whole TDLN (left) and T cell depleted TDLN (right). Representative of 3 individual experiments.