Historically, concomitant tumor immunity has been described for hosts bearing immunogenic tumors, including tumors from outbred animals (1
), methylcholanthrene-induced tumors (4
), and virally transformed models (36
). This response was shown to be complex, with immunity decaying when primary tumors reached large sizes (3
). The work of North et al. ~20 yr ago carefully characterized the immune response to Meth A fibrosarcoma as an equilibrium between tumor-induced CD8+
effector and CD4+
suppressor T cells (3
). Nevertheless, concomitant immunity has not been characterized for poorly immunogenic tumors of spontaneous origin. Resistance to a second inoculum of Madison lung carcinoma and Lewis lung carcinoma has been examined (20
), but immunological mechanisms were not directly implicated, and it remained unclear if growth of such tumors could prime host immunity.
In addition to extending this phenomenon to a poorly immunogenic tumor, the present description of concomitant immunity to B16 melanoma demonstrates possible fundamental differences between strongly and poorly immunogenic tumors. Concomitant immunity to B16 does not occur as a result of progressive tumor growth alone, but rather only as a result of progressive tumor growth in the absence of CD4+ suppressor T cells. These suppressors come from naturally occurring CD4+CD25+ regulatory T cells present de novo in the naive host and are capable of blocking concomitant immunity at both priming and effector phases. GM-CSF production by otherwise poorly immunogenic tumor cells can induce resistance to a second tumor inoculum, although this phenomenon is likely to occur by mechanisms other than simply overcoming suppression, such as enhancing APC recruitment and/or functionality. This supposition is supported by the observation that depletion of CD4+ suppressors further augments the immune response in mice bearing B16-GMCSF primary tumors. Growth of primary B16-GMCSF tumors is strongly dependent on the presence of CD4+ T cells, with CD4 depletion resulting in CD8-dependent rejection of even primary tumors. Therefore, B16-GMCSF may require a basal level of suppressor T cell function to evade immune recognition.
Before this paper, concomitant tumor immunity models had not been characterized with regard to antigen specificity. In the B16 model, we demonstrate that progressive tumor growth elicits immunity to melanocyte differentiation autoantigens. The number of individual CD8+
T cells recognizing any single epitope was modest compared with what might be achieved through active vaccination, and certainly much less than observed after immunity to viruses or bacteria. However, the gp10025
epitopes have been described previously as B16 tumor rejection antigens (24
), and it is most likely that the entire population of CD8+
T cells recognizing multiple shared tumor antigens mediates the potent concomitant immunity that we observe. Furthermore, the presence of cross-reactive immunity to a different melanoma, but not to nonmelanomas, supports the argument that rejection antigens include shared melanocyte differentiation antigens. The fact that JBRH melanoma is not completely rejected suggests that immunity is also directed against antigens specific to B16 and that B16-specific CD8+
T cells are required for complete tumor rejection.
Because these experiments were terminated within ~3 wk due to primary tumor size, we were not able to further characterize autoimmunity (manifested as hypopigmentation of coat) or immunological memory. Memory T cell responses against melanoma differentiation autoantigens have been characteristically difficult to obtain through vaccination (unpublished data). Surgical resection of primary tumors may facilitate future studies of autoimmunity and memory by prolonging the lifespan of tumor-bearing hosts. Postsurgical tumor immunity has been described for highly immunogenic tumors, in many cases with potency exceeding concomitant immunity (20
). This comparison remains to be defined for a poorly immunogenic tumor.
Unexpectedly, priming of concomitant immunity in immune competent mice was only partially reduced in the absence of CD4+ T cell help. However, this result may reflect the regeneration of small numbers of CD4+ T cells between antibody depletions because RAG1−/− mice lacking CD4+ T cells were not able to mount concomitant immunity. Alternatively, local help may be provided by inflammation at the tumor site, and the lack of immunity in RAG1−/− hosts may reflect a difference in antigen-presenting capabilities between these two strains.
Depletion of CD25+
cells by treatment with PC61 mAb has been shown previously to break immunological unresponsiveness to highly immunogenic and, to a lesser extent, poorly immunogenic tumors (41
). PC61 pretreatment of naive mice has been reported to significantly reduce growth of B16 tumors by mechanisms that appeared to involve combinations of NK cells and CD8+
T cells (15
) or CD4+
T cells (41
); however, it remains unclear if relevant CD8+
populations were nonspecifically activated. Our model of concomitant immunity illustrates that, in addition to CD4+
T cell elimination, progressive tumor growth is required for generation of tumor-specific CD8+
T cells and tumor immunity.
In our hands, pretreatment of mice with PC61 mAb was associated with variable growth of primary tumors and CD4+
T cell–mediated rejection of ~20–40% of control tumors, which made it difficult to assess the role of primary tumor growth in rejection of secondary tumors (unpublished data). In addition, treatment with PC61 mAb at later time points was ineffective presumably because of the depletion of activated CD8+
). Rather than PC61, we used GK1.5 mAb, DTA-1 mAb, and cyclophosphamide as tools to block or deplete suppressor T cells. We were interested in DTA-1 mAb because it has been shown previously to block CD4+
suppressor T cell function in vivo (12
). However, more recently, in an allogeneic bone marrow transplant setting, DTA-1 mAb has been shown to act directly on both effector CD4+
T cell populations (43
). Therefore, although our data are collectively consistent with the notion that DTA-1 acts to inhibit CD4+
suppressor T cells, potential activation of effector cells is possible, and further experiments are needed to determine which cell populations are responding to DTA-1 in the B16 concomitant immunity model.
Cyclophosphamide has been described previously to deplete suppressor T cells induced by progressive Meth A tumors and other immunogenic tumors (23
). More recently, a selective reduction in the proportion of CD4+
T cells has been reported in spleens of cyclophosphamide-treated rats (31
). We have observed a similar decline in this population in mice on days 4–10 after cyclophosphamide treatment, although other T cell populations are also affected (unpublished data). Because cyclophosphamide had profound effects on concomitant immunity when administered 4 d before the primary tumor, this evidence supports the role for cyclophosphamide through a mechanism of selective toxicity against CD4+
T cells. However, effects on T cell homeostasis are likely, and further studies are required to examine the exact mechanism of cyclophosphamide function.
The Meth A sarcoma has been shown previously to induce a suppressor population beginning after day 9 of tumor growth (3
), and it has been shown recently that human melanoma can be recognized by a line of CD4+
regulatory T cells derived from the tumor (44
). In addition to suppressor T cell populations, myeloid cells and soluble molecules (e.g., IL-10 and TGF-β) have been shown to contribute to suppression of tumor immunity. Although our studies do not rule out the possibility that B16 growth also generates a CD4+
regulatory T cell population, suppressive CD4+
T cells could not be transferred from mice bearing day-12 tumors. In fact, CD4+
T cells taken from these mice appeared to be inefficient suppressors compared with their naive counterparts. This suggests that tumor growth primes a population of activated CD4+
T cells that participates in tumor immunity, implying that CD4+
T cells isolated from tumor-bearing mice contain a mixed population with different functions. The fact that CD4+
T cells from tumor-bearing mice did not significantly enhance tumor rejection also supports the conclusion that T helper cells were activated and, therefore, were present in the CD4+
population. In any case, CD4+
T cells from naive mice were potent suppressors in vivo after adoptive transfer, without requiring preexposure to tumor. The antigen specificity of the relevant CD4+
T cells within this population remains to be determined, but the finding that regulatory T cell expansion is driven by presentation of self peptides (45
) presents the intriguing possibility that these cells may recognize melanocyte differentiation antigens. In this case, precursor regulatory T cells might not require a priori activation by tumor because these antigens are already expressed by normal cutaneous melanocytes in hair follicles.
One fundamental characteristic of concomitant immunity to tumors and pathogens is the observation that progression of primary tumors or lesions is unaffected by the developing host immune response. For instance, in the case of concomitant immunity to Leishmania major
T cells resident in the primary lesion prevent local immune reactions, while maintaining antigen persistence for priming of systemic immunity (46
). In contrast, concomitant immunity to B16 only proceeds in the absence of preexisting host suppressor cells and, therefore, the presence of a local suppressive environment involving CD4+
T cells only within primary but not challenge tumors is unlikely. Furthermore, significant numbers of tumor-reactive, specific CD8+
T cells are present in lymph nodes draining primary tumors, suggesting that primary tumors can be vulnerable to host immunity under the right circumstances. Indeed, we have observed decreased growth of primary tumors in mice with certain types of concomitant immunity, particularly in those treated with DTA-1 mAb and in T cell–reconstituted RAG1−/−
mice lacking only the CD4+
population. DTA-1 mAb might activate effector cells in addition to inhibiting suppressors, whereas in the case of RAG1−/−
recipients, emerging T cell homeostasis could skew the response strongly to tumor immunity. Although participation of local CD4−
suppressors (such as tumor-associated macrophages) cannot be ruled out, our observation that most primary tumors continue to grow in the face of rejection of challenge tumors likely reflects the fact that these tumors are established with stroma and vasculature before CD8 immunity is primed and, therefore, are more formidable targets for host immunity compared with tumor cells given on day 6. In summary, these studies illustrate the requirements for generating concomitant immunity to a progressive, poorly immunogenic tumor. Our finding that B16 tumors rapidly immunize hosts that lack the CD4+
regulatory T cell population demonstrates the central role played by these cells in the blockade of tumor immunity in this melanoma model. The fact that this concomitant immune response recognizes melanocyte differentiation antigens is consistent with an important role for shared, unaltered self antigens in rejection of poorly immunogenic, spontaneous tumors.