In our present study we found that, contrary to what was previously thought, there is a lack of Ag-dependent specificity of tumor-reactive T cells at the level of infiltration and proliferation in the tumor. Tumor-reactive T cells infiltrate and proliferate in Ag-expressing and nonexpressing tumors in comparable numbers. However, tumor-reactive CD8+ T cells clearly mediate the regression of bulky, established tumor only if it expresses the target Ag.
Although the egress of activated CD8+
T cells into the tumor is not Ag specific (7
), this infiltration may be required for tumor destruction (2
). Thus, workers have attempted to enhance T cell migration to the tumor. In an effort to circumvent the need for trafficking, direct intra-arterial infusion of T cells into the tumor can be successful (19
), but is impractical in the case of widely metastatic disease. In nonaccessible tumor sites, induced expression of adhesion molecules, such as chemokine receptors, integrins, or selectins, may enhance T cell migration to the periphery (20
). The induction of CXCR2 or CXCR4 into T cells may augment T cell targeting to melanoma or leukemias derived from marrow stromal cells that express their respective ligands, CXCL1 or stromal cell-derived factor-1 (23
). Other approaches include modulation of the tumor environment to induce T cell infiltration (25
). However, tumor-reactive T cell migration to the tumor alone does not reliably result in tumor regression (26
). This may be due to the tumor's inability to efficiently activate tumor-reactive T cells (29
), although this may not be the case in all tumors (33
). Thus, a potent T cell activation may be required to mount an effective immune response (14
Exogenous cytokine administration and, in some cases, Ag in the context of danger signals have been shown to stimulate adoptively transferred tumor-reactive T cells and enable them to induce tumor regression (1
). Transfer of these cells into a lymphopenic environment may accentuate these results (our unpublished observations) (10
). The mechanisms involved in this tumor destruction have not been clearly delineated. Although it has been demonstrated that secondary stimulation of CD8+
T cells results in rapid proliferation, a peak, and subsequent contraction in lymphoid organs (15
), little has been shown in peripheral organs. In the current study we found that in vivo stimulation of tumor-reactive T cells not only induces similar kinetics in spleen, blood, and lymph nodes, but also extends into multiple peripheral tissues. This rapid kinetic curve appears to occur simultaneously in all tissues, countering the idea of lymphoid proliferation and then peripheral migration. In addition, we found that several days after stimulation, tumor-reactive T cells were actively dividing in both Ag-positive and negative tumors. This phenomenon is in congruence with several recent studies demonstrating T cell programming and proliferation after a single antigenic exposure (36
). It is interesting to note that active T cell proliferation in the blood was not observed; perhaps some property of or in the peripheral tissues, but not the blood, is necessary for induction into S phase.
Although the migration and proliferation of these in vivo stimulated T cells are not tissue specific, tumor recognition and destruction are specific. IFN-μ
is a reliable indicator of CD8+
T cell-specific target recognition (38
) that induces the up-regulation of MHC I on B16 (16
). Furthermore, cytolytic molecules, such as perforin, can be observed in B16, but not MCA-205, treated with in vivo-stimulated pmel-1 T cells. The release of perforin in conjunction with granzymes by tumor-reactive T cells induces an apoptotic cascade in target tumor (40
), mediating tumor cell death. This tumor killing is visualized by the staining of active caspase 3 in B16 infiltrated with pmel-1 T cells, but not in MCA-205, even in the presence of pmel-1 T cells.
The ability to raise large numbers of tumor-reactive T cells and induce ubiquitous T cell migration and the specific killing of tumor cells by T cells may have important clinical implications. The current vaccination strategies available can induce large quantities of tumor Ag-specific T cells, but these vaccinations do not reliably induce tumor regression (41
). An alternative strategy, based on adoptive T cell transfer, involves the ex vivo expansion of large quantities of tumor-reactive T cells. This methodology obviates the requirement for continuous in vivo vaccination and enables the possibility of pretransfer lymphodepletion, which may enhance T cell-based immunotherapies. Furthermore, an increase in cell number simplifies the logistics involved in genetic modification of T cells with TCRs against tumor Ags (42
) or with homing markers (23
Tumor-homing strategies are currently being explored in our laboratory and elsewhere, but the molecular bases of directing T cells specifically to tumor metastases remain largely unknown. As we have reported in the present manuscript, the in vivo stimulation of adoptively transferred cell circumvents the need for specific targeting, because cells migrate indiscriminately and ubiquitously. In the case of metastatic disease, tumor can infiltrate multiple organ systems. The ability to induce ubiquitous T cell infiltration, while maintaining specific-tumor killing, may be beneficial in the treatment of metastatic, noninflammatory tumor foci.