CCL21 & Tumor Immune Evasion: R&D Systems

Tumor development and growth depends on the ability of tumor cells to evade the host's immune system. Tumor cells employ strategies to impede anti-tumor immune responses, including secretion of immunosuppressive factors and activation of negative regulatory pathways.¹ Recently, Shields et al. reported that invasive tumor cells secrete CCL21.² This group now describes a novel mechanism by which CCL21-secreting tumors transform their outer layer into lymphoid-like tissue to create a tolerant stromal microenvironment that promotes immune system evasion.³

Shields et al. generated mouse melanoma cell sublines expressing different amounts of CCL21 using lentiviral expression and small hairpin RNA (shRNA).³ Murine-derived B16-F10 melanoma cells were transduced with lentivirus carrying a secondary lymphoid tissue-expressed form of murine CCL21 cDNA (CCL21-Ser/CCL21a),^4,⁵ murine CCL21 shRNA, or a scrambled shRNA as a control. CCL21 secretion by these cells was either up-regulated (CCL21^high) or down-regulated (CCL21l^ow), respectively, or remained unchanged (control). The melanoma cell sublines were transplanted into immune competent syngeneic mice. Upon examination of the resulting tumors, the authors found that CCL21-secreting tumors (control and CCL21^high) grew significantly larger than CCL21low tumors. Further investigation revealed that the microenvironments of CCL21-secreting tumors had been reorganized to resemble lymphoid-like tissue.³

Lymphoid tissues are composed of a highly organized network of stromal cells that bring foreign antigens and immune cells into close contact to initiate adaptive immune responses. The formation of lymphoid tissues is coordinated by lymphoid tissue-induced (LTi) cells, which promote the localized expression of chemokines. LTi cells stimulate lymphoid stromal cells, such as fibroblast-like reticular cells (FRCs), to release chemokines that recruit antigen-presenting dendritic cells and lymphocytes into the lymphoid tissue.⁶ FRCs secrete CCL19 and CCL21, stimulating the recruitment of CCR7⁺ cells, such as naïve and memory T cells, mature dendritic cells, and LTi cells.^6,^7,^8,^9,¹⁰ The microenvironments of CCL21-secreting tumors displayed characteristics of lymph tissue. LTi cells were present in the microenvironments of both control and CCL21high tumors, but were not associated with tumors expressing low levels of CCL21.³ In addition, the lymphoid stroma-associated markers, gp38 and ER-TR7, and the high endothelial venule marker, peripheral node addressin (PNAd), were expressed in the stroma surrounding CCL21⁺ tumors, but were absent from the periphery of CCL21^low tumors.³

Cells in the Tumor Microenvironment Promote Immune System Evasion
Cell Type		Proposed Role in Tumor Immune Tolerance
	LTi Cell	Initiates reorganization of the tumor microenvironment
	FRC	Forms the lymphoid-like stroma surrounding the tumor Expresses Crry
	MDSC	Expresses high levels of iNOS
	nTreg/iTreg Cell	Inhibits immune cells Secretes TGF-beta 1
	Dendritic Cell	Expresses IDO
	Tumor Cell	Expresses IDO Expresses Crry Secretes TGF-beta 1
Cells Present in the Microenvironments of CCL21-Secreting Tumors Promote Immune Tolerance. Tumor cells secrete CCL21 and recruit CCR7⁺ cells that restructure the tumor microenvironment to resemble the stroma of lymph tissue. The cells present in the new microenvironment use a variety of mechanisms to promote immune tolerance and facilitate tumor growth. Naturally-derived Treg (nTreg) and tumor cells secrete TGF-beta 1, which suppresses T cell function and converts naïve T cells into induced Treg (iTreg) cells.¹² Tumor and dendritic cells express IDO, the rate-limiting enzyme in tryptophan catabolism. IDO suppresses T cell functions by depleting tryptophan stores and increasing local concentrations of the pro-apoptotic, tryptophan breakdown product, N-formylkynurenine.¹³ Tumor and fibroblast-like reticular cells (FRCs) express Crry, a membrane-associated complement regulatory protein that protects tumor cells from the complement system by inhibiting C3/C5 convertase activity. Both IDO and Crry facilitate tumor growth.^14,¹⁵ Myeloid-derived suppressor cells (MDSCs) express inducible nitric oxide synthase (iNOS), which produces high concentrations of nitric oxide (NO). NO reduces lymphocyte reactions and promotes tumor cell proliferation by inducing the expression of angiogenic factors.¹⁶

Though it seems counterproductive for tumors to create a peripheral environment that is similar to immune tissue, lymphoid tissue can promote immune tolerance. For example, lymph node stromal cells present peripheral tissue antigens to circulating T cells to induce peripheral tolerance.¹¹ Thus, it is plausible that the newly created lymphoid-like stromal structures aid tumor cells in immune system evasion and consequently, enhance tumor growth. Shields et al. uncovered several potential mechanisms that CCL21-secreting tumor cells may utilize to induce immune tolerance.³ CCL21-secreting tumors recruited more CD11b⁺CD11c^–F4/80–Gr1^high myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Additionally, two factors that facilitate tumor growth, indoleamine 2,3-dioxygenase (IDO) and complement receptor 1-related gene/protein y (Crry), were expressed by CCL21-secreting tumors, but were not present in CCL21^low tumors.³ CCL21^low tumors also recruited more mature and cytotoxic T cells specific for melanoma antigen, and had elevated levels of IFN-gamma, IL-2, and IL-4, cytokines associated with anti-tumor immunity and cytotoxic T cell responses. This research gives new insight into how tumor cells influence the immune system by inducing changes in their microenvironment, and provides new avenues for possible cancer treatments, including inhibition of CCL21 secretion by tumor cells or suppression of LTi cell function.