The pro-inflammatory yet immunosuppressive tumour microenvironment is a dynamic structure comprised of stromal, myeloid and lymphoid elements. Changes in microenvironmental cell populations during anti-tumour responses are not well defined. We have developed a flow cytometric method that allows us to identify fifteen distinct murine cell populations within subcutaneous tumours. These include stromal (fibroblastic and endothelial), myeloid (neutrophils, eosinophils, dendritic cells, monocytes and macrophages) and lymphoid cells (B cells, CD4 and CD8 T cells, Tregs, NK, NKT and γδ T cells).
To analyse the antigen-presenting function of myeloid cells isolated from the tumour microenvironment, we used a subcutaneous B16 melanoma expressing Hen-Egg-Lysozyme-Moth-Cytochrome-C (HELMCC) model protein and MCC-specific 5C.C7 TCR-transgenic CD4 T cells. On a per cell basis, tumour DCs induced the greatest proliferation of T cells, followed by macrophages (both Ly6C positive and negative). Neutrophils and eosinophils induced a small amount of proliferation consistent with their expression of low levels of MHCII.
To understand how changes in the tumour microenvironment are related to immune-mediated tumour rejection or progression, we adoptively transferred 5C.C7 CD4 T cells with or without 5C.C7 Tregs into immunodeficient RAG-/- mice bearing B16.HELMCCC tumours. Tumour rejection was associated with an accumulation of neutrophils and IFNg/TNFa/IL-2/Granzyme-producing 5C.C7 T cells in the tumour tissue and a decrease in monocytes and DCs. Tumour progression in Treg recipients was associated with suppression of 5C.C7 CD4 T cell proliferation, effector differentiation and entry into the tumour, and was accompanied by reduced neutrophil influx, and increased eosinophils and macrophages.
Our results provide a new avenue for analysis and specific targeting of tumour populations during immunotherapy. The molecular mechanisms of tumour rejection mediated by CD4 T cells are currently under investigation.