Abstract B49: Autologous T cell responses against patient-derived colorectal cancer xenografts.
Authors: Chou Jeffrey , Mortales Christie-Lynn L. , Ojeaburu Sheila , Yeung Raymond SW , Warren Edus H. 
Published: August 2014

Prognosis in colorectal cancer (CRC) correlates closely with the density of tumor infiltrating T lymphocytes (TIL) in the primary tumor, suggesting that the adaptive cellular immune system can influence the natural history of this disease. However, it is unclear if TIL in CRC comprise a non-specific inflammatory response, or if T cells in TIL are specifically reactive with antigens expressed on CRC tumor cells. To date the study of TIL interaction with autologous CRC cells has been hampered by the small numbers of tumor cells and TIL that can be obtained from CRC surgical samples. We have developed reliable methods for expanding primary human CRC tumor cells as xenografts in immune-deficient mice. Moreover, we have adapted next generation DNA sequencing of T cell receptor β chain CDR3 regions to define the clonal composition of CRC TIL with exquisite precision and depth. In combination with standard procedures for expanding T cells in vitro, we are using these techniques to dissect TIL-CRC tumor cell interactions at the cellular and molecular level.

The CD4+/CD8+ surface phenotype and TCRβ CDR3 sequence repertoire of TIL from 7 CRC tumor samples (a primary colon tumor from one patient, an ovarian metastasis from a second patient, and 4 non-contiguous hepatic metastases from a third patient), and from T cells in blood or normal adjacent tissue was evaluated by flow cytometry and next-generation sequencing, respectively. Flow cytometry revealed that CD4+ T cells comprised the majority of TIL expanded from the tumors from all 3 patients. Comparison of the TCRβ CDR3 sequence repertoires in the tumor and normal tissues demonstrated that the TIL were clonally distinct from the T cells present in blood or normal adjacent tissue. Moreover, extensive overlap in the repertoire of TIL isolated from the 4 hepatic metastases in patient 3 was observed, suggesting the presence in all 4 metastases of a population of T cells with similar antigenic specificity. We designed TCRβ CDR3 sequence-specific PCR primers to determine whether specific TCRβ sequences identified by next-generation sequencing were primarily found in the CD4+ or CD8+ subsets of TIL. The most common CDR3 sequences observed in TIL from the primary colon tumor of patient 1 were primarily found in the CD4+ subset, while the most common CDR3 sequences in the ovarian metastasis from patient 2 and the hepatic metastases from patient 3 were primarily found in the CD8+ subset.

The reactivity of CD8+ and CD4+ T cells expanded from CRC tumor, tumor-associated ascites fluid, normal adjacent tissue, and blood against autologous CRC tumor cells (freshly harvested from xenografts) and autologous dermal fibroblasts was assessed for in a series of 5 patients, including patients 1-3. T cells expanded from the 4 tissue compartments were evaluated by interferon-γ (IFN-γ) ELISPOT for reactivity with xenograft-derived autologous tumor cells or autologous dermal fibroblasts. No significant IFN-γ reactivity was seen when T cells from any compartment were cultured with autologous dermal fibroblasts. In all 5 patients, IFN-γ reactivity with autologous human epithelial cell adhesion molecule (EpCAM) sorted tumor cells was observed. This reactivity was more prominent in purified CD4+ T cells than in CD8+ T cells. No significant difference in the reactivity of T cells from various compartments against autologous tumor cells was observed, but T cells expanded from tumor associated ascites fluid showed overall lower levels of reactivity against all target cells.

Collectively, these results demonstrate that CRC TIL comprise a clonally distinct population of T cells containing both CD4+ and CD8+ T cells that are specifically reactive with autologous CRC tumor cells. However, T cells reactive with autologous tumor cells can also be isolated from other compartments such as normal adjacent tissue and peripheral blood.

Citation Format: Jeffrey Chou, Christie-Lynn L. Mortales, Sheila Ojeaburu, Raymond SW Yeung, Edus H. Warren. Autologous T cell responses against patient-derived colorectal cancer xenografts. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B49.