Role of IL-4 in Inducing Immunosuppressive Tumor Microenvironment in Follicular Lymphoma
Authors: Rawal Seema , Park Hyun Jun , Chu Fuliang , Zhang Min , Nattamai Durga , Kannan Shibi Chakravarthy , Sharma Rakesh , Delgado David A , Chou Tina , Davis Richard Eric , Neelapu Sattva S. 
Published: October 2019

Abstract
Abstract 771

Background:
Recently, using proteomic profiling of tumor lysates, Calvo et al (Blood, 2008) found that IL-4 levels were significantly higher in follicular lymphoma (FL) tissues than in tissues from follicular hyperplasia. IL-4 present in the tumor microenvironment may promote the survival and growth of tumor B cells and may play a role in the pathogenesis of FL. However, the source of IL-4 production was not evaluated in these studies. Several reports have shown that regulatory T cells (Tregs) that can inhibit the function of antitumor CD4+ and CD8+ T cells are increased in number in the tumor microenvironment in FL. Tregs can be induced from conventional CD4+ T cells by the tumor cells but may also be actively recruited to the tumor site by CCL17 and CCL22 produced by tumor cells. However, B cells are not known to constitutively express either of these chemokines. As IL-4 is increased in the FL tumor microenvironment and can activate STAT6, and CCL17 and CCL22 are STAT6-responsive chemokine genes, we tested the possibility that these are interrelated.

Methods:
Surgical tumor samples from FL patients were processed into a single-cell suspension and cryopreserved for later use. Tumor cells isolated by magnetic cell separation were cultured in the presence or absence of IL-4 and chemokine levels were assessed in the supernatants after 48 hours by ELISA. Intracellular cytokine assay was performed on intratumoral T cells and tumor cells to determine the source of IL-4. Western blotting and phosflow was performed to assess the phosphorylation status of STAT6. Migration of Tregs in response to tumor supernatants was assessed in a transwell chemotaxis assay.

Results:
Using primary human FL samples we demonstrate that IL-4 is secreted by intratumoral follicular helper T cells (Tfh) and more importantly, IL-4-producing CD4+ T cells were increased in number in the FL tumor microenvironment as compared with benign follicular hyperplasia. While FL tumor cells produced CCL22 constitutively, the production of CCL17 was induced by IL-4 via activation of STAT6. Consistent with this, we observed a direct correlation between IL-4 and CCL17 mRNA transcripts in whole genome microarray studies of FL tissues (r = 0.41, P = <0.0001). The expression of CCL17 by tumor B-cells was dependent on STAT6 since knockdown of STAT6 using siRNA inhibited its production. IL-4 also stimulated CCL17 secretion from tumor cells from other B-cell lymphomas such as diffuse large cell lymphoma, mantle cell lymphoma, and splenic marginal zone lymphoma as well as intratumoral T cells in FL. CCL17 produced by these cells induced preferential migration of Tregs and IL-4–producing T cells than IFN-g–producing T cells. Antibodies to CCL17 and CCR4 significantly abrogated the migration of Tregs in response to tumor supernatants.

Conclusions:
Our results suggest that IL-4 produced by Tfh cells in FL induces CCL17 production through autocrine and paracrine effects on intratumoral T cells and tumor cells, respectively, and facilitates active recruitment of regulatory T cells and IL-4–producing T cells that in turn may stimulate more CCL17 production. Thus, IL-4 creates a vicious cycle and leads to an immunosuppressive tumor microenvironment that promotes immune evasion and tumor survival and growth. Our results provide novel insights into the role of IL-4 in immunosuppression in FL and other B-cell lymphomas and offer potential targets for development of therapeutic strategies to overcome immune evasion in these malignancies.

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Disclosures:
No relevant conflicts of interest to declare.