<jats:title>Abstract</jats:title> <jats:p>Background: MHC class II-restricted CD4+ T cells reactive against the Id peptide of MHC class II-negative MOPC315 murine myeloma cells can protect mice against injected tumors. This protection occurs via indirect recognition of secreted tumor-specific antigen, presented on MHC class II-molecules of tumor-infiltrating CD11b+ cells. Activated Id-specific Th1 cells produce IFNγ that activate macrophages which become cytotoxic to tumor cells. We wanted to assay the ability of such CD4+/M1 macrophage mediated protection to prevent the outgrowth of antigen-loss tumor variants. Methods: Id-specific TCR-transgenic SCID mice were challenged with a mixed population of Id-secreting and non-secreting MOPC315 cells, differentially labeled with fluorescent proteins. Tumor growth was monitored by in vivo imaging. Tumor-infiltrating macrophages were assayed for their ability to inhibit tumor growth. Results: In the presence of a large excess of antigen-secreting tumor cells, outgrowth of antigen loss-variants occurred with no evidence of bystander killing. A predominance of alternatively activated macrophages was seen in areas dominated by antigen loss-variants, whereas classic activation was the dominant phenotype in areas populated by regressing Id-secreting tumor cells. In vitro, growth of antigen loss-variants was completely inhibited by the provision of the tumor-specific antigen. This data suggests strict spatial requirements of antigen for effective macrophage-mediated protection.</jats:p>