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.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.