Citations

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 Ligand–receptor complexes play a central role in mediating a range of processes in immunology and cancer biology. The ability to directly quantify the fraction of receptors occupied by a ligand in a given biospecimen, as opposed to assessing the concentration of ligand and receptor separately, could provide an additional and valuable clinical and research tool for assessing whether receptors are occupied by a ligand. To address this need, a biomarker platform was developed to quantify the fraction of receptors occupied by a ligand using pairs of RNA aptamers, where one aptamer binds preferentially to the unoccupied receptor and the other to the ligand–receptor complex. Bound aptamer was quantified using RT-qPCR colorimetric probes specific for each aptamer. The binding ratio of aptamer correlated with the fraction of receptors occupied by a ligand. This assay, termed as LIRECAP (LIgand–REceptor Complex-binding APtamer) assay, was used to determine the fraction of soluble CD25 occupied by IL2 in the serum from subjects with B-cell lymphoma. No correlation was found between the type of lymphoma and total soluble CD25 or IL2 independently. In contrast, the fraction of soluble CD25 occupied by IL2 was significantly higher in follicular lymphoma patient serum compared with diffuse large B-cell lymphoma patient serum. We conclude that this technology has the potential to serve as a high-throughput biomarker platform to quantify the fraction of receptors occupied by a ligand.

Key Points Cytokines in the BM microenvironment regulate PD-1 ligand expression and secretion in WM. Secreted PD-1 ligands modulate T-cell function in WM.

Abstract BACKGROUND Monocytes and macrophages (mo/mΦ) are part of composition of tumor microenvironment in B-cell non-Hodgkin lymphoma (B-NHL). CD14+HLA-DRlow monocytes are immunosuppressive and this phenotype is promoted by increased expression of IL-10 in lymphoma. Despite their association with poor outcome, monocytes retain phagocytic function particularly in the presence of monoclonal antibodies and can be associated with an improved outcome in patients treated with rituximab. The aim of this study was to determine the prevalence of CD14+HLA-DRlow mo/mΦ in B-NHL tissue compared to normal tissue, and to determine whether their phenotype suggests that they are capable of phagocytic function. METHODS Tissue mo/mΦ were isolated by negative selection from 13 diagnostic B-NHL biopsy specimens and 6 normal tissues using a monocyte enrichment kit. Morphological and immunophenotypic characteristics of isolated mo/mΦ were determined by flow cytometry, immunocytochemistry and cytometry by time-of-flight (CyTOF). The purity of isolated tissue mo/mΦ was more than 95%, being confirmed by Giemsa stain and flow cytometry. RESULTS Increased numbers of CD68+ cells in initial B-NHL biopsies when compared to normal tissue was confirmed by immunohistochemistry and flow cytometry. However, CD14 expression was substantially lower than CD68 expression in the tissues suggesting that many CD68+ mo/mΦ are CD14 negative. Using a monocyte enrichment kit to isolate all mo/mΦ, we found that CD14 negative mo/mΦ constituted half the tissue mo/mΦ. Furthermore, we found by flow cytometry, CyTOF and Giemsa stain that CD14- mo/mΦ constituted 2 populations: a more frequent population of larger cells and a less common population of smaller cells. While all cells expressed CD68, the larger cells had increased expression of CD32, CD64 and HLA-DR. The CD14+ cells typically also expressed CD163 and CD33 and were a subset of the larger monocyte population, while the population of small cells was positive only for CD68 and CD45. Using CD14 and SIRP-α, we could identify 3 populations of mo/mΦ: CD14+SIRP-αhigh, CD14-SIRP-αlow and CD14-SIRP-α- cells. CD14+SIRP-αhigh cells and CD14-SIRP-αlow cells typically constitute the population of larger cells, while CD14-SIRP-α- cells constituted the population of smaller cells. Interestingly, while the CD14-SIRP-α- cells lack the typical phenotypic markers of mo/mΦ, they morphologically have the appearance of monocytic cells and this population appears expanded in lymphoma tissue. CONCLUSIONS We have identified a unique population of small mo/mΦ that have an immature phenotype and lack expression of CD14, SIRP-α, CD163, and other FcγR markers. This subset of mo/mΦ is prevalent in lymphoma and may have limited phagocytic function. This CD68+CD14-SIRP-α- mo/mΦ subpopulation may account for prognostic differences in the outcome of lymphoma patients treated with or without monoclonal antibodies. Citation Format: Ya-Ping Chen, Zhi-Zhang Yang, Jose C. Villasboas, Tammy Price-Troska, Anne J. Novak, Stephen M. Ansell. Expression of CD14 and SIRP-α defines distinct populations of intratumoral monocytes/macrophages in B-cell non-Hodgkin lymphomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3287.

Key PointsThe NF-κB subunits p52 and RelB increase IRF4 promoter activity and expression in PTCL cells. A positive feedback loop involving CD30, NF-κB, and IRF4 drives PTCL cell proliferation and can be blocked by NF-κB inhibitors.

Abstract Purpose: Transformation of follicular lymphoma is a critical event associated with a poor prognosis. The role of the tumor microenvironment in previous transformation studies has yielded conflicting results. Experimental Design: To define cell subtypes associated with transformation, we examined tissue specimens at diagnosis from patients with follicular lymphoma that later transformed and, using immunohistochemistry (IHC), stained for CD68, CD11c, CD21, CXCL13, FOXP3, PD1, and CD14. Cell content and the pattern of expression were evaluated. Those identified as significantly associated with time to transformation (TTT) and overall survival (OS) were further characterized by flow cytometry and multicolor IHC. Results: Of note, 58 patients were analyzed with median TTT of 4.7 years. The pattern of PD1+ and CD14+ cells rather than the quantity of cells was predictive of clinical outcomes. On multivariate analysis, including the follicular lymphoma international prognostic index score, CD14+ cells localized in the follicle were associated with a shorter TTT (HR, 3.0; P = 0.004). PD1+ cells with diffuse staining were associated with a shorter TTT (HR, 1.9; P = 0.045) and inferior OS (HR, 2.5; P = 0.012). Multicolor IHC and flow cytometry identified CD14+ cells as follicular dendritic cells (FDC), whereas PD1+ cells represented two separate populations, TFH and exhausted T cells. Conclusion: These results identify the presence of PD1+ T cells and CD14+ FDC as independent predictors of transformation in follicular lymphoma. Clin Cancer Res; 20(11); 2862–72. ©2014 AACR.

Key Points STAT5 is constitutively phosphorylated in malignant B cells obtained from patients with Waldenström's macroglobulinemia. Inhibition of STAT5 signaling significantly decreases IgM production and may be useful therapeutically for patients with high IgM levels.