In this study, we investigated the T cell response to a systemically disseminated B cell lymphoma model and found that iNKT and type II NKT cells are critical regulators of the antitumor CD8+ T cell response. We show that in the absence of iNKT cells, lymphoma cells expand in numbers at early time points but appear to be rejected 8–10 days after transplant. This rejection involves CD8+ T cells and allows for long-term tumor free survival in Jalpha18−/− mice. CD8+ T cell responses to OVA-expressing lymphoma cells were vigorous in mice lacking only iNKT cells, were largely reduced in mice containing iNKT cells, and could be further limited in WT mice by the iNKT cell activating ligand alpha-GalCer. The suppressive effects of iNKT cells were observed only in the context of tumor growth and did not reduce CD8+ T cells responses to antigens in the absence of the tumor. Furthermore, our experiments with CD1d1−/− mice suggest that type II NKT cells positively affect the anti-tumor CD8+ T cell response. In the absence of both iNKT and type II NKT cells, the tumor growth and the anti-tumor CD8+ T cell response are comparable to WT mice. Thus, in this system, immunity is mediated at least in part by tumor-specific CD8+ T cells, and the activity of these cells is regulated by iNKT and type II NKT cells.
Similar iNKT versus type II NKT cell antagonism has been noted before 
, but our data differ from the published literature in several important ways. First, although iNKT – type II NKT cell counter-regulation has been posited in tumor systems, ours is the first report where iNKT cells are suppressive to anti-tumor immunity. Most significantly, when iNKT cell-mediated suppression is relieved, the anti-tumor response is sufficient to fully reject a tumor that is universally lethal in WT mice. It is unclear why this lymphoma initiates such a strong suppressive response by iNKT cells, although we hypothesize that several aspects that are unique to lymphoma may play a role.
iNKT cell mediated immunosuppression in lymphoma may reflect the localization of tumor growth within the secondary lymphoid organs, or may simply reflect the fact that the lymphoma developed through transformation of a lymphoid cell and is thus able to manipulate the immune response using mechanisms that are unavailable to other non-immune cell types. The identification of iNKT cell suppression of the CD8+
T cell response to lymphoma is in agreement with previous work demonstrating that iNKT cells are suppressive in several autoimmune models such as rheumatoid arthritis, experimental autoimmune encyphalomyelitis (EAE) 
, and type I diabetes 
(reviewed in 
). iNKT cell suppression of antitumor CD8+
T cell responses is consistent with these observations, as many tumor-associated antigens are self antigens. Because the B cell lymphoma used in the current study arose from an autoreactive B cell 
, it may still elicit the same suppressive pathways used to limit autoreactivity among normal B cells. Thus, the TBL lymphoma may simply be eliciting the immunosuppressive effects of iNKT cells that would normally occur during an autoreactive B cell response. Data from several autoimmune diseases suggests that non-Hodgkin’s lymphomas such as BL often arise from autoimmune settings 
, and correlative evidence suggests that in human non-Hodgkin’s lymphoma, the preneoplastic state may present as an autoimmune polyclonal lymphoproliferative disease 
. Lymphoma arising from these autoinflammatory conditions may be programmed such that the tumor activates the suppressive effects of iNKT cells much more strongly than other tumor types while minimizing the anti-tumor functions of iNKT cells elicited by many solid tumors. Immunization at the time of lymphoma transplant was able to overcome iNKT cell mediated suppression, suggesting that manipulation of the cytokine environment during early tumor growth can alter or overcome the effects of iNKT cells.
The data here present an apparent paradox: that the tumor grows at the same rate whether or not CD8+
T cells are present, thus CD8+
T cells do not affect the growth of the tumor in normal mice. However, in the absence of iNKT cells tumor growth is severely inhibited by the action of CD8+
T cells. How can CD8+
T cells have a detectable role in one situation and not the other? We interpret these data as indicative of the effectiveness of iNKT cell mediated immunosuppression. In normal animals, the iNKT cells somehow completely inhibit the response of CD8+
T cells, therefore the absence of the latter cells in normal animals is not detectable. When inhibition by iNKT cells is absent, the CD8+
T cells are freed to expand and act on the tumor cells. Previous studies have shown that tumors originating from WT mice can grow equally well when transplanted into either WT or Rag2−/−
, suggesting that the processes of immunoediting can produce a tumor that is independent of CD8+
T cell control. We hypothesize that in the TBL system used here, rather than being selected for lower antigenicity through immunoediting, the TBL lymphoma escapes CD8+
T cell control through the induction of iNKT cell mediated immunosuppression.
Studies in a variety of both spontaneous and transplantable solid tumor models 
have combined to establish a role for iNKT cells in activating anti-tumor immunity, primarily through activation of NK and CD8+
T cells 
. Because, to date, no appropriate markers can be used for the unequivocal identification of type II NKT cells, these cells have been less well studied than iNKT cells, and knowledge of their function is largely inferred from comparisons of phenotypes in Jalpha18−/−
mice. The majority of data from solid tumor models suggests that type II NKT cells suppress the anti-tumor response 
. The mechanism for this suppressive effect is generally thought to be mediated by inducing a Th2 cytokine bias through secretion of cytokines such as IL-4 and IL-13. However it is also apparent that other unknown mechanisms, distinct from altering in the Th1/Th2 cytokine balance, might also be involved 
. In contrast to their suppressive functions in solid tumors, our data suggest that type II NKT cells are activating in the anti-lymphoma immune response. Development of better techniques for type II NKT cell identification and function would greatly aid in understanding their function the cancer microenvironment.
While the role of iNKT cells as activators of antitumor immunity has been well documented for solid tumors, evidence that they function similarly in B cell lymphomas is lacking, although there are published examples 
. In contrast, our data suggest an opposite role for iNKT cells in modulating the immune response to B cell lymphoma; that iNKT cells suppress anti-tumor immunity. The suppression of anti-lymphoma immunity by iNKT cells was previously observed using the RMA/S T cell lymphoma, but was in that case dependent on experimentally increasing expression of CD1d on lymphoma cells, which lead to a modest increase in overall survival of CD1d−/−
mice compared to WT mice following tumor challenge 
. Our data illustrate a much more potent effect of iNKT cell loss, where the majority of iNKT cells remain tumor free while all WT mice develop lethal tumors. Suppressive effects of iNKT cells in autoimmunity are generally thought to be caused by production of the Th2 cytokines IL4 and IL-13, while their immunostimulatory effects in other tumor systems are typically IFNγ-dependent (reviewed in 
). It follows that the simplest hypothesis for the iNKT cell dependent CD8+
T cell suppression in the TBL model is the secretion of large amounts of IL-4 and IL-13, which biases the immune system away from a cytotoxic Th1 response. However, administration of alpha-GalCer to tumor bearing mice enhances the ability of iNKT cells to suppress, despite the fact that alpha-GalCer induces strong expression of both IFN-gamma and IL-4 from iNKT cells.
Ours and previously published data 
suggests that, like conventional CD4+
T cells and type II NKT cells, iNKT cells have the capacity to suppress anti-tumor immunity. This iNKT cell-mediated suppression may have been predicted based on the established ability of type II NKT cells to perform this same function 
. Because iNKT cells and type II NKT cells share many common phenotypic and functional features 
, it is not entirely surprising that, under varying conditions present in different tumor microenvironements, both NKT subtypes can perform similar suppressive functions. Our data suggests a role reversal for type II NKT cells. While type II NKT cells generally suppress anti-tumor immune responses 
, the data presented here suggests that they are also capable of supporting the anti-tumor response. In the TBL lymphoma model, type II NKT cells appear to be necessary for effective anti-tumor immunity, but the effects of these cells is limited by the dominant, suppressive effects of iNKT cells. Alternatively, changes to the TCR repertoire of conventional alpha-beta-T cells in Jalpha18−/−
mice could account for the tumor rejection seen in this strain. Disruption of normal TCRalpha gene segment utilization in Jalpha18−/−
mice (that is independent of iNKT cells) may alter composition of the T cell compartment such that the balance of the antitumor response is shifted away from immunosuppression and tumor growth and toward CD8+
T cell mediated rejection. Thus alterations in the conventional T cell repertoire could alternatively explain the tumor phenotype differences observed between Jalpha18−/−
mice, as the latter strain also lacks iNKT cells but has a conventional alpha-beta-T cell repertoire that is comparable to WT mice.
The direct function of T cells in controlling human BL is unclear. The occurrence of immunodeficiency associated BL suggests that the adaptive immune system is important in preventing or suppressing lymphomagenesis, but there is little direct evidence of T cell control of human BL. Although T cells are thought to be important for graft-versus-leukemia and graft-versus-lymphoma (GVL) effects following allogeneic stem cell transplantation, evidence for a significant GVL effect for Burkitt’s lymphoma is incomplete 
. This is likely due in part to the efficacy of current aggressive treatment regimens (reviewed in 
), which greatly reduce the need for additional treatment strategies.
Understanding the cellular elements that regulate anti-tumor responses has the potential to reveal new targets for tumor immunotherapy. Our analysis of the T cell response to a transplanted B cell lymphoma identified NKT cells as critical regulators of the anti-tumor CD8+ T cell response. Determining why some cancers induce immunosuppressive NKT cell phenotypes (mediated by either iNKT or type II NKT cells) while other cancers may induce predominantly anti-tumor NKT cell effects is a major unanswered question in the tumor immunology field. Understanding the function of NKT cells, particularly iNKT cells, in the lymphoma environment is important in designing tumor immunotherapy whether or not the therapy is specifically targeting iNKT cells. Experimental analysis of iNKT cell function in lymphoma rather than solid tumor growth is lacking, and the data presented here suggest a pro-rather than anti-tumor function for this cell type. Our results identify iNKT cell depletion or inhibition as a potential immunotherapy target for Burkitt’s lymphoma.