We have described an in vivo model to assess the role of IL-10 in the regulation of an immune response leading to autoimmunity, as well as protective immune responses. By using the human IL-2 promoter to drive mouse IL-10 cytokine expression, transgene-derived IL-10 produced only transiently in response to T cell activation. At the same time, these transgenic mice have an increased population of T cells that make IL-10 and should produce IL-10 earlier in the immune response. We predicted that activated Th1 type T cells in the IL-10 transgenic mice would downregulate their own activity, including IL-10 secretion from the transgene. Consistent with our expectations, polyclonally activated splenic T cells from transgenic mice secreted two- to fourfold increased levels of IL-10, as well as decreased levels of IFN-γ. The IL-10 transgenic mice were otherwise macroscopically normal, and did not exhibit any defects in growth and development. The relatively subtle alterations in cytokine production observed after polyclonal T cell activation in the transgenic mice may reflect the activity of naive T cells, which normally are capable of making only low amounts of IL-2 (15
). These subtle alterations, however, led to a dramatic decrease in the ability to mount certain immune responses under several circumstances described below.
Consistent with their overall good health and development, the immune system of the IL-10 transgenic mice is by most measures not significantly different from the control littermates. T and B cell numbers and phenotype were normal. Although IL-10 is a potent downregulator of Th1 cytokine production, IFN-γ synthesis is not eliminated in the IL-10 transgenic mice. No major differences were apparent in the serum Ig profiles either. We therefore conclude that the amount of IL-10 synthesized in these mice is not sufficient to cause either a complete suppression of Th1 cytokines or immune dysfunction in unimmunized mice.
It is well documented that transfer of the CD4+
T cell population of normal mice into SCID or RAG-2−/−
mice causes severe inflammation in the large intestine, mediated in part by secretion of TNF-α and IFN-γ (21
). We have shown that transfer of this same population of T cells from the IL-10 transgenic mice does not cause the wasting condition that accompanies this disease. Histological analysis confirmed that the recipients of IL-10 transgenic T cells have few of the hallmarks of intestinal inflammation which are typical of this model. We hypothesize that production of IL-10 alters cytokine secretion from the CD4+
T cells, and thereby inhibits their ability to cause disease. When CD4+
T cells from the IL-10 transgenic mice were co-transferred with the corresponding pathogenic population from control animals, colitis pathogenesis was prevented. These data suggest that the altered IL-10 secretion by the transgenic T cells can control the production of inflammatory cytokines by other cells. In other studies, IL-10−/−
mice spontaneously developed colitis (28
), and transfer of unfractionated lamina propria lymphocytes from IL-10−/−
mice into RAG-2−/−
recipients gave rise to severe inflammation of the colon (29
). Together, these data indicate that T cell– derived IL-10 is critical for regulating the cytokine balance in the intestine that may lead to or prevent colitis pathogenesis.
Our data are consistent with results from a previous report in which transfer of CD4+
cells into SCID mice was accompanied by daily administration of recombinant IL-10 (21
). This treatment prevented the development of colitis and reduced the amount of IFN-γ in the colon. However, the effects were short lived since these mice developed colitis indistinguishable from controls 4 wk after the last injection of rIL-10. The transient nature of the effect of r
IL-10 treatment could be due to the systemic distribution and rapid metabolism of the cytokine. These problems would not have been expected in the transgenic mouse model we describe here, and consistent with this, disease was not observed in SCID recipients of IL-10 transgenic T cells up to 3 mo after transfer. In contrast to these results, it was shown that co-transfer of CD4+
cells into SCID mice, along with anti–IL-10 blocking antibody, did not abolish the protective effect of CD4+
T cells (22
). Because, in our model, Th1-produced IL-10 was sufficient to convert pathogenic T cells to active inhibitors of colitis in co-transfer, we conclude that although the CD4+
T cells may use IL-10 to suppress colitis, other IL-10–independent pathways exist in CD4+
T cells that may be capable of preventing disease.
Control of L. major
infection in mice is mediated by IFN-γ–producing Th1 cells that promote a cell-mediated immune response. IL-10 transgenic mice on the healing C57BL/6 genetic background developed Th1 responses to L. major
that were comparable to those in resistant B10.D2 mice. Thus, IL-10 secretion in these transgenic mice neither skewed cytokine production from T cells, nor inhibited cell-mediated immune responses to the parasite. It is not known why Th1 type cell-mediated responses are diminished in the colitis model but not in response to L. major
infection. The intracellular forms of the parasite induce high levels of IL-12 (30
), and the amount of transgene-derived IL-10 produced under the control of the IL-2 promoter may not be sufficient to overcome the activation of Th1 cells by the pathogen. Reconstitution of SCID mice with L. major
T cells conferred susceptibility to disease that could be reversed by anti–IL-4, but not anti–IL-10 antibodies (22
), consistent with studies in both wild-type (31
) and IL-4–deficient mice (32
). Although IL-10 can inhibit Th1 development and proliferation, IL-4 is critical for the differentiation of Th2 cells (33– 36). The lack of elevated IL-4 levels in the IL-10 transgenic mice after infection presumably underlies the inability of the transgenic mice to develop progressive disease. In summary, the data are consistent with previous information indicating that IL-10 alone is not a key regulator of host responses to L. major.
Although tumors may express antigens, they often escape the host's immune surveillance. It is well known that tumor cells have the capacity to mediate immunosuppression, in part, through the elaboration of immune inhibitory cytokines (25
). Not only may tumors directly suppress immune responses, but they may also induce host immune cells to release immunosuppressive cytokines. For example, human NSCLC cells have been shown to cause a 10–100fold induction of lymphocyte-derived IL-10 (37
). Thus, because the IL-10 transgenic mice used in these studies may parallel the apparent defect in response to lung cancer in human patients, we hypothesized that these mice would have a limited capacity to control tumor growth. For these studies we used an immunogenic murine lung cancer, 3LL. This tumor has a mutant connexin 37 gap junction protein, and a peptide containing the mutated sequence is recognized by anti-3LL CTL (27
). Antitumor CTL are protective against 3LL tumor challenge, suggesting that the cytotoxic responses of CD8+
T lymphocytes are important in limiting tumor growth. To determine if lymphocyte-derived IL-10 interfered with the host antitumor response, IL-10 transgenic and control mice were challenged with 3LL tumor cells. IL-10 transgenic mice were unable to control tumor growth, whereas control recipients showed tumor progression at a significantly slower rate. The inability of the IL-10 transgenic mice to limit 3LL tumor growth is due to the secretion of IL-10, as administration of anti–IL-10–blocking antibody reduced tumor growth in the IL-10 transgenic mice to that of control mice. These data demonstrate that the effect of the IL-10 transgene is due directly to IL-10 action. Previously, only viral, but not mouse, IL-10 was shown to be effective in preventing immune responses to melanomas and sarcomas (38
). There are several ways in which altered expression of mouse IL-10 may have inhibited an effective antitumor immune response. These include the reduction of MHC class I expression (39
) or costimulatory molecules (6
), immune deviation (40
), and induction of anergy in responding T cells (40
). Further experiments are required to characterize the mechanism for tumor unresponsiveness in IL-10 transgenic mice, and to determine if other Th1 or CD8+
T cell responses are likewise suppressed in these transgenic mice.
IL-10 previously has been implicated in the regulation of a wide variety of inflammatory and other immune responses (3
), but many of the studies have relied upon the unphysiologic administration of exogenous IL-10. The transgenic mice described here provide an important new model for studying the role of T cell–derived IL-10 synthesis in vivo. These mice have been backcrossed extensively onto the C57BL/6 background, so that the IL-10 transgenic mice and their littermates are close to being isogenic strains. Furthermore, IL-10 synthesis is transient and related to T cell activation, thereby avoiding any potential toxic effects of either constitutive expression of IL-10, or very high levels of IL-10 over expression. Finally, evidence from two models, a pathogenic immune response leading to colitis and a beneficial immune response leading to tumor rejection, demonstrates that some, but not all, cell-mediated immune responses are profoundly altered in these transgenic animals. Recently, IL-10 transgenic mice that express cytokine under the control of the CD2 promoter have been described (43
). Consistent with the results described here, these mice show some evidence of a suppressed Th1 type response, as they are more susceptible to infection with Mycobacterium tuberculosis.
Additional experiments now underway are designed to determine the mechanism for decreased colitis pathogenesis and tumor cell rejection in the IL-10 transgenic mice.