Hirashima, Mitsuomi

Abstract Abstract 2668 Poster Board II-644 Interleukin-12 (IL-12) has been demonstrated to induce IFN-g production by T and NK cells and thereby contribute to anti-tumor immunity. However, the administration of IL-12 to boost anti-tumor immunity in B-cell lymphoma has shown no clinical benefit. In fact, clinical trials of IL-12 in combination with rituximab in follicular B-cell lymphoma (FL) showed a lower response rate in patients treated with the combination than in patients treated with rituximab alone (Clin Cancer Res. 2006 15; 12:6056-63). The goal of this study was therefore to determine the role of IL-12 in the antitumor response in B-cell NHL. First, we measured serum levels of IL-12 in patients with untreated FL before treatment with rituximab and normal healthy controls. We found that serum IL-12 levels were elevated in FL patients compared to healthy individuals (median: 0.50 ng/ml, n=30 vs 0.32 ng/ml, n=22; p= 0.03) and that elevated serum IL-12 levels were associated with a poor outcome in these patients when treated with rituximab alone as initial therapy. Using 0.56 ng/ml as a cutoff, patients with serum IL-12 levels of greater than 0.56 ng/ml had a significantly shorter time to progression than patients with IL-12 levels less than 0.56 ng/ml (12 months versus 40 months; p=0.001). To determine the mechanism by which IL-12 may contribute to a poor prognosis, we investigated the role of IL-12 on induction of immune tolerance. First, we found that TIM-3, a member of the T cell immunoglobulin and mucin domain-containing protein (TIM) family that functions to terminate TH1-mediated immunity and promote tolerance, was constitutively expressed on a subset of intratumoral T cells accounting for approximately 15% and 25% of the intratumoral CD4+ and CD8+ T cells, respectively. In contrast, less than 2% of T cells from peripheral blood of normal individuals expressed TIM-3. TIM-3-expressing T cells were distinct from regulatory T cells since CD25+ and Foxp3+ T cells lacked TIM-3 expression. Secondly, we found that TIM-3-expressing CD4+ cells were unable to produce cytokines such as IL-2, IFN-g or IL-17 and that TIM-3-expressing CD8+ T cells failed to produce Granzyme B, IFN-g or IL-2. We also observed that TIM-3-expressing T cells lost the capacity to proliferate in response to TCR activation. These results suggest that TIM-3 expressing CD4+ and CD8+ T cells are functionally exhausted. Thirdly, we observed that TIM-3 expression on T cells could be induced by activation and that IL-12 was the strongest stimulus to induce TIM-3 expression on CD4+ and CD8+ T cells. Finally, we found by immunohistochemistry (IHC) that Galectin-9 (Gal-9), a ligand for TIM-3, was abundantly expressed on lymphoma B cells. In vitro incubation with a stable form of Gal-9 induced apoptosis of CD4+ and CD8+ T cells in a dose dependent fashion. Gal-9-mediated apoptosis of T cells was attenuated by a TIM-3 Fc protein and isolated TIM-3+ T cells exhibited a significantly higher apoptosis rate than TIM-3− T cells in response to Gal-9. These results indicate that, in contrast to the observations in vitro or in vivo in mice, IL-12 actually plays a detrimental role in lymphoma patients. Given the findings that IL-12 strongly induces TIM-3 expression on effector T cells and that the TIM-3/Gal-9 pathway impairs the immune response, we conclude that increased serum levels of IL-12 suppress anti-tumor immunity in follicular lymphoma patients and is associated with a poor prognosis. Disclosures: Witzig: Novartis: Research Funding.

Abstract Abstract 143 IL-12 induces IFN-g production and contributes to anti-tumor immunity. However, administration of IL-12 in cancer patients has resulted in a limited clinical benefit. In follicular B-cell lymphoma (FL), a clinical trial of IL-12 in combination with rituximab showed a lower response rate in patients treated with the combination than in patients treated with rituximab alone (Clin Cancer Res 2006), suggesting that, in contrast to the observations in vitro or in vivo in mice, IL-12 actually plays a detrimental role in FL patients. Recently, a type of immune response, termed “ T cell exhaustion” describing a condition in which T cells exhibit reduced differentiation, proliferation, and effector function, has been shown to impair anti-tumor immunity and result in disease progression. TIM-3, a family member of T-cell immunoglobulin and mucin domian-containing proteins, inhibits TH1-mediated immune response and promotes immunological tolerance. A recent study has suggested that TIM-3 may play an important role in mediating T cell exhaustion. However, the biological and clinical relevance of TIM-3 in cancers remains completely unknown. In this study, we determined whether T cell exhaustion exists in the tumor microenvironment, whether IL-12 contributes to T cell exhaustion, and whether TIM-3-mediated T cell exhaustion impacts patient outcome in FL. We found that serum IL-12 levels were elevated in FL patients compared to healthy individuals (median: 0.50 ng/ml, n=30 vs 0.32 ng/ml, n=22; p= 0.03) and that elevated serum IL-12 levels were associated with a poor outcome in these patients when treated with rituximab alone as initial therapy. Using 0.56 ng/ml as a cutoff, patients with serum IL-12 levels of greater than 0.56 ng/ml had a significantly shorter time to progression than patients with IL-12 levels less than 0.56 ng/ml (12 months versus 40 months; p=0.001). Both lymphoma B cells and monocytes were able to produce IL-12 and contributed to elevated serum levels of IL-12 in FL. Importantly, we found that IL-12 strongly induced TIM-3 expression in a dose-dependent manner. Endogenous production of IL-12 by lymphoma B cells and monocytes was capable of regulating TIM-3 expression on T cells from FL. As a consequence, TIM-3 was highly expressed on a subset of T cells from PBMCs or lymph nodes from FL patients while its expression was negligible or moderate on T cells from normal PBMCs or benign lymph nodes, respectively. The number of TIM-3-expressing T cells accounted for approximately 32% and 39% of CD4+ or CD8+ T cells in biopsy specimens and 6.2% and 6.7% in peripheral blood of FL patients. TIM-3+ T cells, co-expressed with PD-1, exhibited a reduced ability to proliferate and decreased cytokine production compared to TIM-3- T cells. Similarly, IL-12-induced TIM-3+ T cells gradually lost the capacity to produce cytokines over a period of time. These results suggest that TIM-3-expressing T cells are functionally exhausted. In addition, TIM-3+ T cells were prone to apoptotic induction by its ligand galectin (Gal) -9. The phosphorylation of p38 was higher in TIM-3+ T cells compared to TIM-3- T cells when exposed to Gal-9, suggesting MAPK pathway was involved in Gal-9-mediated apoptosis of TIM-3+ T cells. Finally, increased numbers of intratumoral TIM-3-expressing cells were associated with a higher histological grade, higher LDH levels and a poor survival in FL patients. Taken together, these results indicate that IL-12, in contrast to its role in augmenting immune response through IFN-g, induces T cell exhaustion by upregulating TIM-3 expression. We further demonstrated that lymphoma B cells produce IL-12 thereby contributing to T cell exhaustion by promoting TIM-3 expression on intratumoral T cells. Impairment of anti-tumor immunity due to T cell exhaustion induced by the IL-12-TIM-3 pathway may account for the observation that high levels of serum IL-12 and increased number of TIM-3+CD4+ T cells correlate with a worse outcome in FL patients. These findings not only reveal a novel IL-12-TIM-3 pathway that plays an important role in impairing tumor immunity and detrimentally affecting prognosis in FL patients, but may have therapeutic potential for cancer patients. Disclosures: No relevant conflicts of interest to declare.

Abstract Ecalectin, produced by Ag-stimulated T lymphocytes, is a potent eosinophil-specific chemoattractant in vitro as well as in vivo and thus is implicated in allergic responses. Ecalectin differs structurally from other known eosinophil chemoattractants (ECAs); ecalectin belongs to the galectin family defined by their affinity for β-galactosides and by their conserved carbohydrate recognition domains. These characteristic features suggest that ecalectin has unique activities associated with allergic inflammation besides ECA activity. Conversely, ecalectin may mediate ECA activity by binding to a receptor of a known ECA via affinity for the β-galactosides present on this receptor. In this study, we have tested whether ecalectin mediates ECA activity by binding to a receptor of a known ECA, and we have assessed its effects on eosinophils. Ecalectin did not mediate ECA activity by binding to the IL-5R or to CCR3. Also, the ECA activity of ecalectin was mainly chemokinetic. In addition, ecalectin induced concentration-dependent eosinophil aggregation, a marker for eosinophil activation. Ecalectin induced concentration-dependent superoxide production from eosinophils but did not induce degranulation; usually these two events are coupled in eosinophil activation. Moreover, ecalectin directly prolonged eosinophil survival in vitro and did not trigger eosinophils to secrete cytokines that prolong eosinophil survival. These results demonstrate that ecalectin has several unique effects on eosinophils. Therefore, we conclude that ecalectin is a novel eosinophil-activating factor. Presumably, these effects allow ecalectin to play a distinctive role in allergic inflammation.