Because of its multiple distinct functions in a variety of experimental models of T cell immunology, it has been difficult to develop a clear model of the in vivo roles of TGF-β[7, 40]. There is ample data to support the hypothesis that TGF-β is an immunosuppressive factor. As summarized previously[3, 8, 15, 16, 40], TGF-β has been reported to (1) inhibit T cell proliferation, CTL generation, and T cell cytokine production; (2) interfere withTH1/TH2 differentiation and the differentiation of naïve T cells towards central memory cells; and (3) inhibit dendritic cell (DC)-mediated antigen presentation by inhibiting DCs’ endocytic and phagocytic activities, preventing DC-maturation, and blocking the up-regulation of critical DC-associated co-stimulatory molecules.
In contrast, there are other studies (although fewer) that have reported that TGF-β exerts stimulatory effects on human T cells and dendritic cells. There is evidence that under some conditions, TGF-β can (1) support the generation of effector cells; (2) augment the development of memory and mature T cell populations; (3) co-stimulate the growth and maturation of CD4+ and CD8+ T cells; (4) inhibit the apoptosis of CD4+ T cells; (5) promote the in vitro development of DCs from hematopoietic progenitors; and (6) regulate the chemotaxis of DCs via regulation of chemokine receptor expression[40–46].
Based on the paradigm that TGF-β is “one of the most potent immunosuppressors described to date”, translational investigators have tried to inhibit tumor growth in animal models by blocking TGF-β production, receptor binding, or function. Using a number of approaches that include anti-TGF-β antibodies, soluble receptors, or TGF-β-binding proteins[6, 17], investigators have consistently reported that blockade of TGF-β is therapeutically useful in a number of murine tumor systems, including renal cell cancer, melanoma, hepatocellular carcinoma, and glioma.
The literature is currently unable to bridge these seemingly contradictory findings regarding TGF-β in cancer biology. The observed results likely depend on the experimental models used, the type of stimulus, the presence of other cytokines, the dose of TGF-β, the distribution of TGF-β in its latent and active form, the duration of the stimulation, and possibly, the genetic background of the cell populations studied. Regardless of the reasons, since TGF-β blocking agents are currently being developed for clinical use, it has become increasingly important to better understand the effects of TGF-β on in vivo anti-tumor immune cell function.
We observed that blockade of TGF-β with sTGF-βR before the inoculation of tumor cells (and for 3 doses afterwards) resulted in significantly enhanced tumor growth of one particular tumor cell line, the AB12 line (but not in others). This response was in marked contrast to the inhibition of tumor growth associated with administration of the same TGF-β blocking agent after the establishment of the same tumor cell line. In this study, we examined the mechanism responsible for the increased rate of AB12 tumor growth resulting from pretreatment with sTGF-βR. We demonstrated that altered anti-tumor immune responses were responsible for this augmentation of tumor growth; specifically, administration of sTGF-βR before tumor cell-inoculation resulted in the failure to generate active anti-tumor CTLs.
The specific characteristics of the relatively immunogenic tumor model used in these studies are important to understand our findings. Mesotheliomas usually result from prior asbestos exposure. They are associated with a high degree of MHC class I expression and TGF-β production. Clinically, they respond to some immune-based therapies. The mouse mesothelioma tumor cells used in this study are very similar to human tumors. When AB12 cells are injected into syngeneic BALB/c mice, their initial growth is quite slow until about 20 days, at which point their size begins to increase rapidly (Figures 2A,2E, and6A). It appears that this initial slow growth phase is due to a partially effective anti-tumor immune response mediated by endogenous, functionally active tumor antigen-specific CTLs. We have observed that AB12 tumors grow much more rapidly in SCID mice (Figure 2F), in CD8+ T cell-depleted mice (Figure 3B), and in IFNγ-knockout or IFNγ-neutralized mice (data not shown). We have also directly examined the ability of AB12 tumors to generate anti-tumor immune responses. Within 4–10 days after subcutaneous injection of AB12 tumor cells, we have detected CD8+ T cells in the spleen that have cytolytic activity. We confirmed the presence of these spontaneously generated anti-tumor CTLs in this study (Figure 4) using a Winn assay that demonstrated markedly inhibited tumor growth when tumor cells were mixed with CD8+ splenocytes from control tumor-bearing animals before inoculation into naïve non tumor-bearing animals. These anti-tumor CTLs persist until the tumor reaches a size of approximately 400 mm3 (usually about 20 days after injection). At this time, CTL activity can no longer be detected and tumor growth rate rapidly increases.
Our experiments indicate that the increased rate of AB12 tumor growth resulting from pretreatment with sTGF-βR was due to a loss of this normal, low-level, and only partially-effective anti-tumor CTL immune response. First, the growth-augmenting effects of sTGF-βR relative to IgG2a were lost in T cell-deficient SCID mice (Figure 2F) and CD8+ T cell-depleted mice (Figure 3B). Second, we showed that the inhibition of TGF-β negatively impacts the functionality of CD8+ CTLs, as the Winn assay (essentially an in vivo test of CD8+ T cell functionality) demonstrated a reduced anti-tumor response with an equivalent number of CD8+ T cells from mice pretreated with sTGF-βR compared to control animals pretreated with IgG2a (Figure 4). Together, these results implicate the inhibition of anti-tumor CD8+ CTLs as central to the augmentation of AB12 tumor growth associated with sTGF-βR pretreatment.
In addition to our tumor study, we also investigated the effect of TGF-β-blockade on the generation of active antigen-specific CTLs against a known viral tumor antigen in an independent and more quantifiable system. Pretreatment with sTGF-βR, at a time point before immunization with an adenovirus encoding the HPV E7 protein (Ad.E7), inhibited the generation of E7-specific CD8+ T cells as compared to control pretreatment with murine IgG2a. These experiments show that TGF-β is required for the generation of active CTLs, at least in models employing AB12 tumor cells or vaccination with Ad.E7.
Unfortunately, despite further investigation, the mechanism by which pretreatment with sTGF-βR inhibits CTL-activity remains unclear. Initial sensitization of CD8+ T cells typically requires 4 steps as described above. We showed that pretreatment with sTGF-βR does not decrease the activation status or the number of DCs, CD4+ T cells, or CD8+ T cells in the TDLNs or tumor beds compared to IgG2a. These data indicate that TGF-β may not be required for the migration or proliferation of DCs, CD4+ T cells, or CD8+ T cells or the activation of DCs. Although studies of expression levels of CD86, MHC class I, and MHC class II are important to evaluate the activation levels of DCs in anti-tumor immune responses, other activation markers for DCs might exist, such as ICAM-1 or B7. It may also be important to test the expression levels of accessory molecules on T lymphocytes, such as LFA-1 or CD28. Thus, the mechanism by which pretreatment with sTGF-βR stimulates the growth of tumors in our AB12 tumor model remains unclear.
Another interesting question relates to the issue of why sTGF-βR did not inhibit the generation of anti-tumor CD8+ CTL activity in other tumor models as it did in the AB12 tumor model. We explored a number of obvious explanations: low amounts of TGF-β produced, lack of tumor immunogenicity, or animal strain differences. With regard to TGF-β production, we know that AB-1 cells make very little TGF β which could explain the lack of effect in this cell line. However, the TC-1 cell line makes sizeable amounts of TGF-β and yet it is still resistant. We have also studied the L1C2 and TC-1 cell lines in the past and have shown them to be moderately or highly immunogenic, similar to the AB12 model, and able to induce anti-tumor CD8+ T cells. To address the issue of strain differences, we also studied L1C2 cells, another tumor line that grows in BALB/c mice (like AB12), and saw no response. We thus have no simple explanation for the selectivity for our observation. The tumor microenvironment is a complex ecosystem which is unique to each tumor model. Given the genetic modifications required for malignant transformation, it is likely that a myriad of factors, including various cytokines, chemokines, other soluble factors, and even cell-bound mediators play significant roles in tumor development and in the interaction with the host’s immune system. The key point is that this stimulation of tumor growth after early TGF-β inhibition can occur in at least one animal model and thus should be carefully looked for in future clinical trials. Additional ongoing research that identifies the key factors responsible for this effect will be needed.