Clinical Advances in Hematology & Oncology
December 2012, Volume 10, Issue 12
Zhi-Zhang Yang, MD, and Stephen M. Ansell, MD, PhD
Dr. Yang is an Assistant Professor and Dr. Ansell is a Professor in the Division of Hematology, Department of Internal Medicine, at Mayo Clinic, in Rochester, Minnesota.
Address correspondence to: Stephen M. Ansell, MD, PhD, Division of Hematology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905; Phone: 507-266-2161; Fax: 507-266-9277; E-mail: email@example.com
Abstract: Like other B-cell lymphomas, the development and progression of follicular lymphoma (FL) involves complex interactions between the neoplastic B cells and the surrounding microenvironment. Malignant B cells can manipulate the microenvironment by skewing the differentiation of immune cells, attracting regulatory T cells or suppressive monocytes, or secreting cytokines that promote an immunosuppressive environment. The importance of the microenvironment in FL has been demonstrated using methodologies such as gene expression profiling, which has shown that the nature of the tumor microenvironment predicts survival in patients with FL and may influence the response to immunotherapy and risk of transformation. Strategies that both enhance an effective antitumor response and reverse immunosuppression and dysfunction will be essential in the development of effective immunotherapeutic approaches in this disease.
Follicular lymphoma (FL), the second most common type of non-Hodgkin lymphoma (NHL), is a serious and often fatal illness.1 The clinical course of this disease is variable, and the molecular and cellular mechanisms responsible for the clinical heterogeneity of follicular B-cell NHL are largely unknown. However, it is becoming increasingly clear that the tumor microenvironment in FL plays an important role in disease severity, clinical outcome, and response to therapy.2-4
The tumor microenvironment is comprised of the normal cells, molecules, and blood vessels that surround and feed a tumor cell. A tumor can change its microenvironment, and the microenvironment can affect how a tumor grows and spreads. The structure and composition of the tumor microenvironment varies among different types of cancers and from patient to patient. For example, the specific structure of secondary lymphoid organs from which most lymphomas originate (eg, the lymph nodes and spleen) makes the tumor microenvironment of hematologic malignancies significantly different from that of solid tumors. There are abundant immune cells in secondary lymphoid organs, which distinguish hematologic malignancies from solid tumors where the immune cells infiltrate in limited numbers. The frequency, distribution, and function of immune cells differ considerably among patients with the same type of cancer, which has been shown to impact patient outcomes. This article will focus on the immune cells and cytokines in the tumor microenvironment of FL.
Composition of Lymph Nodes
In most patients with FL, the lymph node is the main organ in which lymphoma cells reside. The microenvironment of FL is complex in terms of structure and cellular composition because the lymph node has a unique architecture and function. Structurally, the lymph node can be divided into 3 compartments: the cortex (outer region), paracortex, and the medulla (inner region). Follicles containing germinal centers (GCs) are located within the cortex. Different types of cells preferentially reside in separate areas within these compartments. B cells are usually found in the follicles within the outer cortex, while T cells are mainly present within the paracortex and medulla. Upon antigen (Ag) stimulation, T and B cells home to T-cell zone and follicles in lymph nodes, interact with Ag-presenting cells (APCs), and undergo clonal expansion. Dendritic cells (DCs) are potent APCs that collect and process Ag from tissues, carry them to the lymph nodes, and present them to T cells to initiate primary immune responses. Follicular dendritic cells (FDCs) are the APCs that present Ag to B cells and are present only in follicles of primary and secondary lymph organs. Follicles with germinal centers are specifically important for normal B-cell maturation. Before Ag confrontation, the follicle is composed of unstimulated, naïve B cells and does not have a germinal center. Upon Ag stimulation, follicles develop germinal centers, where B cells differentiate and become immunoglobulin-secreting plasma cells. Although most T cells reside outside of the follicle, an important subset of CD4+ T cells known as follicular T helper (TFH) cells reside specifically within germinal centers and are responsible for Ag-dependent activation of B cells in the follicle. CD8+ T cells are rarely seen in the germinal center of follicles. Scattered macrophages are sparsely present inside the germinal center and are responsible for removing debris from apoptotic cells.
In FL, the structural architecture and cellular composition within follicles differs from normal lymph nodes. While follicles are kept intact in FL, the germinal centers become larger due to increasing numbers of lymphoma B cells. An altered cellular composition is commonly seen in biopsies of FL when compared to that of normal lymph nodes. For example, increased regulatory T (Treg) cells and decreased effector T cells, such as TH1 and TH17 cells, are found in FL. Many of the effector T cells present in the tumor microenvironment of FL have an exhausted phenotype and exhibit limited function. In addition, immature immune-suppressive macrophages are present in increased numbers in FL. These alterations have all been shown to potentially affect antitumor immunity and support lymphoma cell survival and growth in FL.
Intratumoral T Cells
Follicular lymphoma is characterized by the presence of a significant number of T cells, up to 50% of the cell mixture, in the tumor microenvironment. These intratumoral T cells have a substantial impact on antitumor immunity and patient outcome.5 T cells are generally heterogeneous and influence tumor immunity both positively and negatively, depending on the prevalence of various T-cell populations within the microenvironment. Generally, elevated numbers of intratumoral T cells are associated with favorable prognosis in patients with FL,6-8 but further studies have found that specific subsets of T cells correlate with patient outcome.9,10
CD8+ T Cells
T lymphocytes can be divided into 2 populations: CD4+ and CD8+ T cells. Generally, CD4+ T cells provide help to other immune cells, and CD8+ T cells are mainly engaged in target cell killing through the secretion of perforin and granzyme B (GrzB). In FL, the frequency of intratumoral CD8+ T cells varies, ranging from 10–50% in different patients when determined by immunohistochemistry.11 Unlike CD4+ cells that are seen both inside and outside the follicles, CD8+ cells are mostly perifollicular. It could be speculated that these perifollicular CD8+ cells protect the follicle; however, 3-dimensional confocal laser scanning microscopy showed that most CD8+GrzB+ cells appeared to target FL B cells, as contact between cytotoxic CD8+ T-lymphocytes (CTLs) and FL B cells was observed.11 Supporting this finding, in vitro-generated CD8+ T-cell clones show specific cytotoxicity directed against autologous lymphoma cells.12-14 Clinically, higher intratumoral CD8+ T-cell numbers correlate with longer overall survival (OS) and disease-specific survival (DSS), independent of the Follicular Lymphoma International Prognostic Index (FLIPI) and all other prognostic factors,11,15 suggesting that these cells are part of a lymphoma-specific immune response.
CD4+ T Cells
CD4+ T cells play a central role in the immune response by helping B cells make antibodies; promoting enhanced antimicrobial activity by macrophages; recruiting neutrophils, eosinophils, and basophils to sites of inflammation; and, through their production of cytokines and chemokines, orchestrating an effective immune response. Since it was recognized that CD4+ cells could be separated into cells that made interferon (IFN)-γ and those that produced interleukin (IL)-4,16 it has been realized that CD4+ T cells are not a single set of cells, but represent a series of cell populations with distinct functions.
Treg Cells A notable advancement in T-cell biology was the identification of CD4+CD25+ Treg cells.17 Treg cells are a small subset of CD4+ T cells expressing CD25 (naturally occurring Treg cells) and represent approximately 5–10% of peripheral CD4+ T cells in both mice and humans. Inducible Treg cells are Treg cells that are generated by antigenic stimulation. It has been demonstrated that the forkhead/winged helix transcription factor family member p3 (Foxp3) is a master transcriptional regulator for the development and function of Treg cells; as such, Foxp3 is now used as a specific marker for Treg cells.18-20 Treg cells were originally identified as able to suppress T cells. Subsequent studies have found that Treg cells are able to suppress other types of immune cells, such as B cells and natural killer (NK) cells, thereby influencing various types of immune responses, including autoimmune, antimicrobial, and antitumor immune responses. Elevated numbers of Treg cells are generally found in the peripheral blood and biopsy specimens of patients with various cancers. In FL, Treg cells are highly represented and have been found to efficiently suppress intratumoral CD4+ and CD8+
T cells, resulting in suppressed antitumor immunity.21-23 The mechanisms accounting for elevated numbers of Treg cells in B-cell NHL include increased recruitment and de novo generation of Treg cells. It has been shown that chemokine receptor 4 (CCR4) and chemokine ligand 22 (CCL22) play a crucial role in attracting Treg cells into the tumor site.21 In addition to recruitment of Treg cells, de novo generation of Treg cells is another important mechanism in FL.24-26 Tumor-derived, transforming growth factor (TGF)-β induces Foxp3 expression and contributes to tumor-mediated conversion of CD4+CD25- T cells into Treg cells.27 Interaction between CD27-CD70 is involved in lymphoma B-cell–mediated generation of Treg cells, as CD70-expressing lymphoma
B cells are efficient in inducing Foxp3 expression in intratumoral CD4+CD25- T cells.24
In addition to biological relevance, Treg cells have also shown significant clinical relevance in FL. By immunohistochemistry, Carreras and associates9 studied a cohort of 97 FL patients and found that Foxp3+ Treg cells were present in all patients, with frequency varying from patient to patient. Low numbers of Treg cells correlated with refractory disease, transformation, and aggressive histology. They noted that high numbers of Treg cells predicted improved survival in FL, a finding that has been supported by other studies.28 However, subsequent studies failed to produce consistent results and found that the number of infiltrating Foxp3 cells did not correlate with overall survival in FL.29,30 Such a discrepancy may be due to heterogeneous therapies administered to patients in these studies. In this regard, Farinha and colleagues30 conducted a study involving 105 advanced-stage FL patients enrolled in a phase II clinical trial who were treated uniformly with multi-agent chemotherapy and radiation. By tissue microarrays, the authors found that Treg cell content did not impact survival, but the distribution pattern of Foxp3+ Treg cells correlated with patient outcome. Patients with Foxp3+ cells localized in a follicular pattern (intrafollicular or perifollicular) displayed a significantly elevated risk of transformation and shorter survival than patients with Foxp3+ cells present in a diffuse pattern, a finding that is supported by other reports.7 Taken together, these results suggest that the architecture of the microenvironment, and particularly the location of Treg cells in the architecture, has an impact on patient outcome in FL.
TH1 and TH2 Cells CD4+ T cells were initially defined as helper cells (TH) in order to distinguish them from cytotoxic CD8+ T cells. Based on their cytokine-secreting profiles, TH cells were initially divided into TH116 and TH216 cells. Along with the recent identification of BCL-6 as a master transcriptional factor for follicular helper
T cells,31,32 the TH family has expanded into 4 major lineages: TH1,16 TH2,16 TH17,33,34 and TFH35,36 cells. CD4+CD25+ Treg cells, as discussed above, form the other major lineage of CD4+ T cells.37 Treg cells and TH cells constitute 2 opposing immune responses and are critically involved in the modulation of immune responses in lymphoma. TH1 cells normally produce cytokines such as IFN-γ, TGF-β, and IL-2, and mediate cellular immune response by enhancing the killing capacity of macrophages and cytotoxic CD8+ T cells. TH2 cells secrete cytokines such as IL-4, IL-5, and IL-6, and mediate humoral immune responses by stimulating B-cell proliferation and antibody production. TH17 cells produce cytokines such as IL-17 and IL-22, and induce inflammation by stimulating inflammatory cytokine production. TFH cells are a subpopulation of CD4+ T cells and specifically help germinal center B-cell maturation and differentiation. Before the addition of TH17 and TFH, studies had been focused on the TH1 or TH2 immune response, as the balance between the 2 is critically skewed in many human diseases. For example, immune responses in acute bacterial infection, such as tuberculosis, are often TH1 type dominant, and inflammation in atopy/allergy, such as asthma, is mediated by TH2 type immunity. In lymphoma, skewing between a TH1 and TH2 phenotype has not been definitively supported by the literature. Jones and coworkers38 investigated 44 B-cell NHL patients and found that both TH1 and TH2 cytokines were expressed at high mRNA levels, measured by reverse transcriptase-polymerase chain reaction (RT-PCR). Although it is generally believed that the TH1 immune response is more effective than TH2 for antitumor immunity, and that TH2 immune response actually favors tumor growth by both promoting angiogenesis and inhibiting TH1 immune response, this study38 observed that high IL-4 levels correlated with longer survival duration. In contrast, our group39 has found that elevated serum levels of IL-12, the major cytokine in TH1 immunity, are associated with a poor prognosis in FL. These findings therefore do not support the traditional concept of TH1/TH2-mediated antitumor immunity; in fact, this concept has been challenged, as the biological effects of TH1 and TH2 cells are often found to be inconsistent with clinical observations.
TH17 Cells Due to the fact that TH17 cells were identified and characterized only recently, data on TH17 cells are limited in FL. The frequency of TH17 cells is low in FL when compared to other types of B-cell NHL,40 and malignant B cells play an important role in suppressing TH17 cell differentiation, thereby leading to the reduced presence of TH17 cells in FL. Costimulatory molecules participate in this suppression because the use of a blocking antibody against CD70, CD80, or CD86 reversed lymphoma
B cell–mediated inhibition of TH17 cells.24 This inhibition also correlated with enhanced Treg cell differentiation.40 The low frequency of TH17 cells and high representation of Treg cells leads to inhibition of inflammation and may account for the lack of an inflammatory immune response in the tumor microenvironment in FL patients.
TFH Cells While the biology of TFH cells has been intensively investigated under normal physiological conditions, TFH cells have not been assessed under pathological circumstances. Because of its specific localization, TFH can be detected in biopsies of FL.39,41 The intratumoral TFH cells express high intensity staining for PD-1 and CXCR5, as well as BCL-6.42 A recent study41 quantified CD4+CXCR5+ICOS+ T cells and found that the frequency of this population was higher in lymph nodes from FL than from diffuse large B-cell lymphoma (DLBCL) or reactive lymph nodes. This population of cells is heterogeneous in terms of CD25 and Foxp3 expression. CD25 and Foxp3 expression divides this population into 2 subsets: follicular T helper (TFH) cells and follicular regulatory T (TFR) cells. Both subsets display unique gene expression profiles. TFH cells support FL B-cell activation and rescue autologous malignant B cells from spontaneous apoptosis.41 In contrast, TFR cells exert a strong regulatory function by inhibiting CD4+CD25- effector T-cell proliferation. Furthermore, purified FL-derived TFH cells overexpress several genes potentially involved (directly or indirectly) in lymphomagenesis (in particular, IL-4 or CD40L), which efficiently rescue malignant B cells from spontaneous and rituximab (Rituxan, Genentech/Biogen Idec)-induced apoptosis.41,43
Factors Regulating T-Cell Function
T-cell function is regulated by many factors, including suppressive signals from costimulatory molecules. One such set of signals is via the PD-1/PD-L1 signaling pathway. Programmed death 1 (PD-1), a member of the CD28/CTLA-4 family, is induced in activated T cells. PD-1, interacting with its receptor PD-L1, has been shown to negatively regulate T-cell receptor (TCR) signaling and decrease proliferation and cytokine production in T cells. We21 observed that PD-1 is highly expressed on intratumoral CD4+CD25- T cells, and that CD4+CD25+ Treg cells express PD-L1 upon TCR activation. This expression pattern suggested a role for PD-1 and PD-L1 interaction in intratumoral Treg cell–mediated immune suppression. In fact, blocking the interaction between PD-1 and PD-L1 with a neutralizing antibody attenuated Treg cell–mediated inhibition of CD4+CD25- T cells. A study of 100 FL samples confirmed the expression of PD-1 on T cells and also found that PD-1 expression was rarely found on Foxp3+ Treg cells.10
TIM-3, a family member of T-cell immunoglobulin and mucin domain proteins, has been shown to inhibit TH1-mediated auto- and allo-immune responses, and to promote immunologic tolerance.44,45 Recently, a growing number of studies have suggested that, instead of functioning as an inhibitor for TH1 cells, TIM-3 actually plays a crucial role in mediating T-cell exhaustion and contributing to negative immune responses in both viral infections and tumors.46-50 We have found that TIM-3–expressing T cells were frequently detected in biopsy specimens of FL patients and displayed exhausted phenotypic and functional characteristics.39 The intratumoral TIM-3+ T cells also express PD-1 and have impaired function. Of note, PD-1high CD4+ T cells in FL biopsies have also been found to co-express CXCR5 and have been characterized as TFH cells, suggesting that TIM-3 may be more specific as a marker for exhausted T cells than PD-1. Importantly, the numbers of CD4+TIM-3+ T cells were associated with a poor survival in FL patients. We found that IL-12 plays a key role in upregulating TIM-3 expression, inducing T-cell exhaustion, and contributing to the high number of TIM-3+ T cells in FL.39
In contrast to TIM-3 expression, the prognostic significance of PD-1 expression has been controversial. In initial studies, the number of PD-1+ cells was significantly lower in patients with a poor performance status and high serum lactate dehydrogenase (LDH), and high numbers of PD-1+ T cells were associated with improved overall survival in FL patients.10,51 However, subsequent studies52 have challenged this finding and have identified PD-1+ T cells as an independent prognostic risk factor for decreased overall survival in FL. The disparate findings may be due to differences in the study populations, patient selection criteria, or clinical management, as well as the varying methodologies used to enumerate PD-1+ T cells. The finding that PD-1+ T cells are heterogeneous cells with phenotypes of both TFH and exhausted T cells may contribute to the discrepant observations.
Intratumoral Monocytes/Macrophage/Dendritic Cells
Cells of the monocyte lineage comprise different types based on maturation status, and their progeny includes macrophages and dendritic cells. Differentiation of these cells is defined by a variety of markers expressed on the cell surface, including CD11c, CD14, and CD68. Monocytic cells play an essential role in the innate immune response as a first line of resistance against pathogens and in activating adaptive immune responses. Depending on stimulation, macrophages may undergo classical M1 activation (stimulated by lipopolysaccharide and IFN-γ) or alternative M2 activation (stimulated by IL-4 and IL-13). The resulting M1 and M2 macrophages produce distinct cytokines and play different roles in the innate immune response. For example, M1 macrophages produce IL-12 and promote TH1 cell development, and M2 macrophages secret IL-10 and facilitate the development of TH2 cells.53 In the early stage of tumors, M1 macrophages are recruited and infiltrate into the tumor microenvironment in response to inflammatory signals, and then release proinflammatory cytokines and chemokines to promote T- and NK-cell development and differentiation. In the later stages of tumor development, macrophages differentiate into a subpopulation called tumor-associated macrophages (TAMs). TAMs may polarize to M2 cells and release cytokines to encourage TH2 differentiation and recruitment. It has been shown that TAMs inhibit antitumor immunity by secreting suppressive cytokines such as TGF-β, by promoting angiogenesis, and by expressing growth factors that support tumor growth.53 Clinically, it has been observed that increased numbers and/or density of intratumoral macrophages correlate with both progression and prognosis in the majority of cancers, including B-cell non-Hodgkin and Hodgkin lymphoma.54 Similar findings have been observed in FL. Farinha and associates55 sought to determine the role of multiple biomarkers in determining outcome in FL, with a specific focus on the role of macrophages. Using tissue microarrays, CD68 staining was the only biomarker that correlated significantly with the overall survival of FL patients. Increased CD68+ cell content, termed lymphoma-associated macrophages (LAM), was associated with diminished survival. This finding supported a previous gene-profiling study in FL that found a macrophage-related immune signature correlated with a poor prognosis.5 Other studies have confirmed that CD68+ LAMs are associated with an adverse outcome, but have also shown that a therapeutic regimen, such as rituximab, can circumvent this association.56,57
Myeloid-Derived Suppressor Cells
Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of immature myeloid cells that have not yet differentiated into macrophages, dendritic cells, or granulocytes, and are normally present in the bone marrow of healthy individuals. In this regard, MDSCs are generally divided into 2 distinct subpopulations: monocytic and granulocytic MDSCs. While it is easy to define MDSCs in mice by using CD11b and Gr-1, the phenotypes to define this population in humans are quite divergent in studies employing different types of tissues. Thus, the lack of consensus in defining MDSCs makes it difficult to quantify this population in patient samples.58,59 Various studies, however, have shown that MDSCs are highly represented in a variety of cancers and suppress NK and T cells through either direct cell contact, cytokines, or byproducts of metabolic pathways. MDSCs have also been shown to regulate expansion and activation of Treg cells, support angiogenesis, and promote tumor cell metastasis.
MDSCs have been intensively investigated in solid tumors; however, few studies have been performed in hematologic malignancies. Recently, Lin and colleagues60 identified a CD14+ subpopulation with low or absent human leukocyte antigen DR (HLA-DR) expression in the peripheral blood of patients with B-cell NHL, including FL. These CD14+DRlow/- monocytes inhibit T-cell function with a mechanism involving arginine metabolism. An elevated frequency of this suppressive monocytic subpopulation correlated with disease progression and overall survival. Studies in multiple myeloma61 and T-cell lymphoma62 support these findings and highlighted the role of HLA-DR expression in defining MDSCs in hematologic malignancies.
Because MDSCs are immature monocytic or granulocytic cells with immunosuppressive properties, it may be beneficial to promote differentiation to mature cells, such as macrophages and dendritic cells, and thereby decrease MDSCs. Thus far, 2 different agents have been shown to facilitate this process: 25-hydroxyvitamin D3 (VD3) and all-trans-retinoic acid (ATRA). Therapeutic administration of either VD3 or ATRA has been found to be associated with decreased numbers of MDSCs and increased numbers of mature HLA-DR+ monocytes in cancer patients.63-65 In agreement with these findings, recent studies found that VD3 insufficiency significantly correlates with a poor outcome in patients with hematologic malignancies.66,67
Other Intratumoral Cells
Natural Killer Cells
NK cells are a type of cytotoxic lymphocyte critical to innate immunity that do not require recognition of the major histocompatibility complex (MHC) presented on infected cell surfaces. NK cells comprise 2 main subsets: less mature CD3-CD56bright and more mature CD3-CD56dim cells, based on functional and phenotypical characteristics.68 CD56bright cells are mainly present in lymph nodes, express dim or negative CD16, and produce abundant cytokines, such as tumor necrosis factor (TNF)-α and IFN-γ. In contrast, CD56dim cells are more commonly found in peripheral blood with bright expression of CD16, and have a cytotoxic function.68 While most studies emphasize the role of intratumoral T cells and macrophages, few studies have focused on NK cells in B-cell NHL. Gibson and coworkers69 analyzed the total number of NK cells, as well as NK subsets, and correlated the number with disease progression in B-cell NHL. They found that the total number of NK cells varied based on tissue site, and the proportions of NK cells were similar based on Ann Arbor stage. However, the proportions of NK cells did correlate with the FLIPI score.69 Data obtained from patients with other B-cell lymphomas found that higher proportions of peripheral blood
NK cells were associated with a favorable prognosis.70,71
Follicular Dendritic Cells
Follicular dendritic cells (FDCs) are the stromal cells located in the GC of follicles. FDCs comprise 1% of all GC cells.72 Fully differentiated FDCs express low-affinity IgE receptor CD23, complement receptors CD21 and CD35, and lymphocyte chemoattractant CXCL13. Unlike other APCs, FDCs do not internalize, process, and present Ag, but present intact Ag-antibody (Ab) complexes on their cell surface. B cells that bind to these Ag-Ab complexes survive and differentiate into memory B cells or plasma cells. In vitro studies73 using FDC cell lines such as HK cells showed that FDCs preferentially bind GC to B cells, providing them with survival signals, while the majority of unbound B cells undergo apoptosis. CD40 ligand and cytokines such as IL-15 are critically involved in FDC-mediated, GC B-cell proliferation and survival.73 In FL patients, FDCs may be immature and show partial or complete absence of surface markers, such as CD23, CD21, or CD35.74-76 FL patients with mature FDCs in the lymph nodes have increased numbers of FL-associated T cells, and the mature FDCs are associated with a higher clinical stage at the time of diagnosis.74 In contrast, Cui and associates77 found that atypical immature FDCs, which have diminished or absent CD21 staining, are present in 11% of FL patients. The majority of patients with a predominance of mature FDCs in the tumor showed advanced clinical stage (III or IV), whereas cases demonstrating an atypical immature FDC network showed localized clinical stage (I or II).77
In addition to the cellular compartment, cytokines (including chemokines) form another molecular compartment that critically influences tumor immunity and patient outcome in cancer. Among the cytokine family, interleukin (IL)-2 and its signaling components have been investigated in B-cell NHL, including FL. IL-2 was originally identified as a T-cell growth factor and was later found to promote the function of a variety of other immune cells, such as B cells, NK cells, and macrophages. Administration of IL-2 to cancer patients achieves a promising outcome, although the duration of benefit is often short.78 Based on experimental and clinical results from other cancers, recombinant IL-2 (rIL-2) has been administered to enhance the efficacy of standard therapy regimens, especially rituximab, in FL patients.79,80 Several clinical trials observed that, although rIL-2 boosts immune responses determined by enhanced NK-cell numbers and antibody-dependent cellular cytotoxicity (ADCC) activity, response rates were low,80 suggesting that enhanced immune responses do not directly translate into meaningful clinical benefit for FL patients. Additional studies have found that IL-2 is essential to the development of Treg cells that suppress tumor immunity, which may account for the limited benefit of rIL-2 in FL patients.
To identify cytokines and cytokine receptors that may be important in FL, our group performed a multiplex ELISA (Luminex) assay on serum specimens obtained from 30 previously untreated patients and compared the levels of 30 cytokines in these patients to those in normal controls. We observed elevated serum levels of a number of cytokines that included soluble IL-2 receptor alpha (sIL-2Rα).81 Higher serum IL-2Rα levels before treatment were associated with a shorter progression-free survival in FL patients treated with rituximab alone as initial therapy, which is consistent with the findings in other aggressive lymphomas.82-84 Biologically, sIL-2Rα, instead of blocking, actually maintains IL-2 signaling and induces Foxp3 expression in T cells, resulting in a regulatory phenotype. These results indicate that sIL-2R plays an active biologic role in FL by binding IL-2 and sustaining IL-2 signaling rather than depleting IL-2 and blocking its function.
In FL, the data collected from in vitro assays or animal models do not consistently translate to clinical benefit. An example is the use of IL-12. IL-12 induces IFN-γ production and promotes the function of T and NK cells, which contributes to antitumor immunity. However, administration of IL-12 to boost antitumor immunity in cancer patients has shown minimal or no clinical benefit.85 In fact, a clinical trial of IL-12 in combination with rituximab in B-cell NHL showed a lower response rate in patients treated with the combination than in patients treated with rituximab alone.86 Supporting this finding, in FL patients, elevated IL-12 levels at diagnosis were associated with an inferior outcome.39 These results suggested that, in contrast to the observations made in vitro or in vivo in mice, IL-12 actually plays a detrimental role in FL patients. Further study has revealed that IL-12 upregulates TIM-3 expression and induces T-cell exhaustion, which accounts for inferior outcome in FL patients treated with IL-12.39
Serum or intratumoral levels of other cytokines, including TGF-β, vascular endothelial growth factor (VEGF), IL-10, and TNF-α, have been shown to correlate with survival in patients with FL.87-89 All of these cytokines regulate immune cell differentiation and function, and may play a role in promoting malignant B-cell growth and survival. Additional research is needed to define the role of intratumoral cytokines, as regulation of cytokine production may present a therapeutic opportunity for patients with lymphoma.
FL has a unique tumor microenvironment compared to solid tumors, and the presence of a substantial number of intratumoral immune cells has been shown to influence antitumor immunity and patient survival. Immune cells, such as T cells and macrophages, can reside within follicles and have direct contact with lymphoma B cells. Malignant B cells can manipulate the microenvironment by skewing the differentiation of immune cells, attracting regulatory T-cells or suppressive monocytes, or secreting cytokines that promote an immunosuppressive environment. All of these mechanisms promote immune suppression and immune exhaustion, as well as immune dysfunction within sites involved by lymphoma, making it difficult for tumor-specific effector cells to kill malignant cells. Therefore, to develop effective immunotherapeutic approaches in lymphoma, we will need to apply strategies that both enhance an effective antitumor response and reverse immunosuppression and dysfunction.
1. Friedberg JW, Taylor MD, Cerhan JR, et al. Follicular lymphoma in the United States: first report of the national LymphoCare study. J Clin Oncol. 2009;27:1202-1208.
2. de Jong D, Fest T. The microenvironment in follicular lymphoma. Best Pract Res Clin Haematol. 2011;24:135-146.
3. Coupland SE. The challenge of the microenvironment in B-cell lymphomas. Histopathology. 2011;58:69-80.
4. de Jong D, Koster A, Hagenbeek A, et al. Impact of the tumor microenvironment on prognosis in follicular lymphoma is dependent on specific treatment protocols. Haematologica. 2009;94:70-77.
5. Dave SS, Wright G, Tan B, et al. Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells. N Engl J Med. 2004;351:2159-2169.
6. Ansell SM, Stenson M, Habermann TM, Jelinek DF, Witzig TE. Cd4+ T-cell immune response to large B-cell non-Hodgkin’s lymphoma predicts patient outcome. J Clin Oncol. 2001;19:720-726.
7. Lee AM, Clear AJ, Calaminici M, et al. Number of CD4+ cells and location of forkhead box protein P3-positive cells in diagnostic follicular lymphoma tissue microarrays correlates with outcome. J Clin Oncol. 2006;24:5052-5059.
8. Wahlin BE, Sundstrom C, Holte H, et al. T cells in tumors and blood predict outcome in follicular lymphoma treated with rituximab. Clin Cancer Res. 2011;17:4136-4144.
9. Carreras J, Lopez-Guillermo A, Fox BC, et al. High numbers of tumor-infiltrating FOXP3-positive regulatory T cells are associated with improved overall survival in follicular lymphoma. Blood. 2006;108:2957-2964.
10. Carreras J, Lopez-Guillermo A, Roncador G, et al. High numbers of tumor-infiltrating programmed cell death 1-positive regulatory lymphocytes are associated with improved overall survival in follicular lymphoma. J Clin Oncol. 2009;27:1470-1476.
11. Laurent C, Muller S, Do C, et al. Distribution, function, and prognostic value of cytotoxic T lymphocytes in follicular lymphoma: a 3-D tissue-imaging study. Blood. 2011;118:5371-5379.
12. Wang J, Press OW, Lindgren CG, et al. Cellular immunotherapy for follicular lymphoma using genetically modified CD20-specific CD8+ cytotoxic T lymphocytes. Mol Ther. 2004;9:577-586.
13. Baskar S, Kobrin CB, Kwak LW. Autologous lymphoma vaccines induce human T cell responses against multiple, unique epitopes. J Clin Invest. 2004;113:1498-1510.
14. Grube M, Rezvani K, Wiestner A, et al. Autoreactive, cytotoxic T lymphocytes specific for peptides derived from normal B-cell differentiation antigens in healthy individuals and patients with B-cell malignancies. Clin Cancer Res. 2004;10:1047-1056.
15. Wahlin BE, Sander B, Christensson B, Kimby E. CD8+ T-cell content in diagnostic lymph nodes measured by flow cytometry is a predictor of survival in follicular lymphoma. Clin Cancer Res. 2007;13:388-397.
16. Seder RA, Paul WE. Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu Rev Immunol. 1994;12:635-673.
17. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol. 1995;155:1151-1164.
18. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol. 2003;4:330-336.
19. Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat Immunol. 2003;4:337-342.
20. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science. 2003;299:1057-1061.
21. Yang ZZ, Novak AJ, Stenson MJ, Witzig TE, Ansell SM. Intratumoral CD4+CD25+ regulatory T-cell-mediated suppression of infiltrating CD4+ T cells in B-cell non-Hodgkin lymphoma. Blood. 2006;107:3639-3646.
22. Hilchey SP, De A, Rimsza LM, Bankert RB, Bernstein SH. Follicular lymphoma intratumoral CD4+CD25+GITR+ regulatory T cells potently suppress CD3/CD28-costimulated autologous and allogeneic CD8+CD25- and CD4+CD25- T cells. J Immunol. 2007;178:4051-4061.
23. Yang ZZ, Novak AJ, Ziesmer SC, Witzig TE, Ansell SM. Attenuation of CD8(+) T-cell function by CD4(+)CD25(+) regulatory T cells in B-cell non-Hodgkin’s lymphoma. Cancer Res. 2006;66:10145-10152.
24. Yang ZZ, Novak AJ, Ziesmer SC, Witzig TE, Ansell SM. CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells. Blood. 2007;110:2537-2544.
25. Ai WZ, Hou JZ, Zeiser R, Czerwinski D, Negrin RS, Levy R. Follicular lymphoma B cells induce the conversion of conventional CD4+ T cells to T-regulatory cells. Int J Cancer. 2009;124:239-244.
26. Mittal S, Marshall NA, Duncan L, Culligan DJ, Barker RN, Vickers MA. Local and systemic induction of CD4+CD25+ regulatory T-cell population by non-Hodgkin lymphoma. Blood. 2008;111:5359-5370.
27. Liu VC, Wong LY, Jang T, et al. Tumor evasion of the immune system by converting CD4+CD25- T cells into CD4+CD25+ T regulatory cells: role of tumor-derived TGF-beta. J Immunol. 2007;178:2883-2892.
28. Tzankov A, Meier C, Hirschmann P, Went P, Pileri SA, Dirnhofer S. Correlation of high numbers of intratumoral FOXP3+ regulatory T cells with improved survival in germinal center-like diffuse large B-cell lymphoma, follicular lymphoma and classical Hodgkin’s lymphoma. Haematologica. 2008;93:193-200.
29. Sweetenham JW, Goldman B, LeBlanc ML, et al. Prognostic value of regulatory T cells, lymphoma-associated macrophages, and MUM-1 expression in follicular lymphoma treated before and after the introduction of monoclonal antibody therapy: a Southwest Oncology Group Study. Ann Oncol. 2010;21:1196-1202.
30. Farinha P, Al-Tourah A, Gill K, Klasa R, Connors JM, Gascoyne RD. The architectural pattern of FOXP3-positive T cells in follicular lymphoma is an independent predictor of survival and histologic transformation. Blood. 2010;115:289-295.
31. Johnston RJ, Poholek AC, DiToro D, et al. Bcl6 and Blimp-1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation. Science. 2009;325:1006-1010.
32. Nurieva RI, Chung Y, Martinez GJ, et al. Bcl6 mediates the development of T follicular helper cells. Science. 2009;325:1001-1005.
33. Harrington LE, Hatton RD, Mangan PR, et al. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol. 2005;6:1123-1132.
34. Park H, Li Z, Yang XO, et al. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol. 2005;6:1133-1141.
35. Schaerli P, Willimann K, Lang AB, Lipp M, Loetscher P, Moser B. CXC chemokine receptor 5 expression defines follicular homing T cells with B cell helper function. J Exp Med. 2000;192:1553-1562.
36. Breitfeld D, Ohl L, Kremmer E, et al. Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production. J Exp Med. 2000;192:1545-1552.
37. Sakaguchi S. Naturally arising CD4+ regulatory t cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol. 2004;22:531-562.
38. Jones EA, Pringle JH, Angel CA, Rees RC. Th1/Th2 cytokine expression and its relationship with tumor growth in B cell non-Hodgkin’s lymphoma (NHL). Leuk Lymphoma. 2002;43:1313-1321.
39. Yang ZZ, Grote DM, Ziesmer SC, et al. IL-12 upregulates TIM-3 expression and induces T cell exhaustion in patients with follicular B cell non-Hodgkin lymphoma. J Clin Invest. 2012;122:1271-1282.
40. Yang ZZ, Novak AJ, Ziesmer SC, Witzig TE, Ansell SM. Malignant B cells skew the balance of regulatory T cells and TH17 cells in B-cell non-Hodgkin’s lymphoma. Cancer Res. 2009;69:5522-5530.
41. Ame-Thomas P, Le Priol J, Yssel H, et al. Characterization of intratumoral follicular helper T cells in follicular lymphoma: role in the survival of malignant B cells. Leukemia. 2012;26:1053-1063.
42. Hilchey SP, Rosenberg AF, Hyrien O, et al. Follicular lymphoma tumor-infiltrating T-helper (T(H)) cells have the same polyfunctional potential as normal nodal T(H) cells despite skewed differentiation. Blood. 2011;118:3591-3602.
43. Pangault C, Ame-Thomas P, Ruminy P, et al. Follicular lymphoma cell niche: identification of a preeminent IL-4-dependent T(FH)-B cell axis. Leukemia. 2010;24:2080-2089.
44. Sanchez-Fueyo A, Tian J, Picarella D, et al. Tim-3 inhibits T helper type 1-mediated auto- and alloimmune responses and promotes immunological tolerance. Nat Immunol. 2003;4:1093-1101.
45. Sabatos CA, Chakravarti S, Cha E, et al. Interaction of Tim-3 and Tim-3 ligand regulates T helper type 1 responses and induction of peripheral tolerance. Nat Immunol. 2003;4:1102-1110.
46. Jones RB, Ndhlovu LC, Barbour JD, et al. Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection. J Exp Med. 2008;205:2763-2779.
47. Jin HT, Anderson AC, Tan WG, et al. Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection. Proc Natl Acad Sci U S A. 2010;107:14733-14738.
48. Fourcade J, Sun Z, Benallaoua M, et al. Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients. J Exp Med. 2010;207:2175-2186.
49. Sakuishi K, Apetoh L, Sullivan JM, Blazar BR, Kuchroo VK, Anderson AC. Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity. J Exp Med. 2010;207:2187-2194.
50. Zhou Q, Munger ME, Veenstra RG, et al. Co-expression of Tim-3 and PD-1 identifies a CD8+ T-cell exhaustion phenotype in mice with disseminated acute myelogenous leukemia. Blood. 2011;117:4501-4510.
51. Wahlin BE, Aggarwal M, Montes-Moreno S, et al. A unifying microenvironment model in follicular lymphoma: outcome is predicted by programmed death-1–positive, regulatory, cytotoxic, and helper T cells and macrophages. Clin Cancer Res. 2010;16:637-650.
52. Richendollar BG, Pohlman B, Elson P, Hsi ED. Follicular programmed death 1-positive lymphocytes in the tumor microenvironment are an independent prognostic factor in follicular lymphoma. Hum Pathol. 2011;42:552-557.
53. Schmieder A, Michel J, Schonhaar K, Goerdt S, Schledzewski K. Differentiation and gene expression profile of tumor-associated macrophages. Semin Cancer Biol. 2012;22:289-297.
54. Bingle L, Brown NJ, Lewis CE. The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies. J Pathol. 2002;196:254-265.
55. Farinha P, Masoudi H, Skinnider BF, et al. Analysis of multiple biomarkers shows that lymphoma-associated macrophage (LAM) content is an independent predictor of survival in follicular lymphoma (FL). Blood. 2005;106:2169-2174.
56. Taskinen M, Karjalainen-Lindsberg ML, Nyman H, Eerola LM, Leppa S. A high tumor-associated macrophage content predicts favorable outcome in follicular lymphoma patients treated with rituximab and cyclophosphamide-doxorubicin-vincristine-prednisone. Clin Cancer Res. 2007;13:5784-5789.
57. Canioni D, Salles G, Mounier N, et al. High numbers of tumor-associated macrophages have an adverse prognostic value that can be circumvented by rituximab in patients with follicular lymphoma enrolled onto the GELA-GOELAMS FL-2000 trial. J Clin Oncol. 2008;26:440-446.
58. Montero AJ, Diaz-Montero CM, Kyriakopoulos CE, Bronte V, Mandruzzato S. Myeloid-derived suppressor cells in cancer patients: a clinical perspective. J Immunother. 2012;35:107-115.
59. Pastula A, Marcinkiewicz J. Myeloid-derived suppressor cells: a double-edged sword? Int J Exp Pathol. 2011;92:73-78.
60. Lin Y, Gustafson MP, Bulur PA, Gastineau DA, Witzig TE, Dietz AB. Immunosuppressive CD14+HLA-DR(low)/- monocytes in B-cell non-Hodgkin lymphoma. Blood. 2011;117:872-881.
61. Brimnes MK, Vangsted AJ, Knudsen LM, et al. Increased level of both CD4+FOXP3+ regulatory T cells and CD14+HLA-DR(-)/low myeloid-derived suppressor cells and decreased level of dendritic cells in patients with multiple myeloma. Scand J Immunol. 2010;72:540-547.
62. Wilcox RA, Wada DA, Ziesmer SC, et al. Monocytes promote tumor cell survival in T-cell lymphoproliferative disorders and are impaired in their ability to differentiate into mature dendritic cells. Blood. 2009;114:2936-2944.
63. Lathers DM, Clark JI, Achille NJ, Young MR. Phase 1B study to improve immune responses in head and neck cancer patients using escalating doses of 25-hydroxyvitamin D3. Cancer Immunol Immunother. 2004;53:422-430.
64. Mirza N, Fishman M, Fricke I, et al. All-trans-retinoic acid improves differentiation of myeloid cells and immune response in cancer patients. Cancer Res. 2006;66:9299-9307.
65. Kusmartsev S, Su Z, Heiser A, et al. Reversal of myeloid cell-mediated immunosuppression in patients with metastatic renal cell carcinoma. Clin Cancer Res. 2008;14:8270-8278.
66. Drake MT, Maurer MJ, Link BK, et al. Vitamin D insufficiency and prognosis in non-Hodgkin’s lymphoma. J Clin Oncol. 2010;28:4191-4198.
67. Shanafelt TD, Drake MT, Maurer MJ, et al. Vitamin D insufficiency and prognosis in chronic lymphocytic leukemia. Blood. 2011;117:1492-1498.
68. Caligiuri MA. Human natural killer cells. Blood. 2008;112:461-469.
69. Gibson SE, Swerdlow SH, Felgar RE. Natural killer cell subsets and natural killer-like T-cell populations in benign and neoplastic B-cell proliferations vary based on clinicopathologic features. Hum Pathol. 2011;42:679-687.
70. Palmer S, Hanson CA, Zent CS, et al. Prognostic importance of T and NK-cells in a consecutive series of newly diagnosed patients with chronic lymphocytic leukaemia. Br J Haematol. 2008;141:607-614.
71. Plonquet A, Haioun C, Jais JP, et al. Peripheral blood natural killer cell count is associated with clinical outcome in patients with aaIPI 2-3 diffuse large B-cell lymphoma. Ann Oncol. 2007;18:1209-1215.
72. Schnizlein CT, Kosco MH, Szakal AK, Tew JG. Follicular dendritic cells in suspension: identification, enrichment, and initial characterization indicating immune complex trapping and lack of adherence and phagocytic activity. J Immunol. 1985;134:1360-1368.
73. Park CS, Choi YS. How do follicular dendritic cells interact intimately with
B cells in the germinal centre? Immunology. 2005;114:2-10.
74. Chang KC, Huang X, Medeiros LJ, Jones D. Germinal centre-like versus undifferentiated stromal immunophenotypes in follicular lymphoma. J Pathol. 2003;201:404-412.
75. Bagdi E, Krenacs L, Krenacs T, Miller K, Isaacson PG. Follicular dendritic cells in reactive and neoplastic lymphoid tissues: a reevaluation of staining patterns of CD21, CD23, and CD35 antibodies in paraffin sections after wet heat-induced epitope retrieval. Appl Immunohistochem Mol Morphol. 2001;9:117-124.
76. Said JW, Pinkus JL, Shintaku IP, et al. Alterations in fascin-expressing germinal center dendritic cells in neoplastic follicles of B-cell lymphomas. Mod Pathol. 1998;11:1-5.
77. Cui W, Che L, Sato Y, et al. Nodal follicular lymphoma without complete follicular dendritic cell networks is related to localized clinical stage. Pathol Int. 2011;61:737-741.
78. Rosenberg SA, Lotze MT, Muul LM, et al. A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high-dose interleukin-2 alone. N Engl J Med. 1987;316:889-897.
79. Dang NH, Fayad L, McLaughlin P, et al. Phase II trial of the combination of denileukin diftitox and rituximab for relapsed/refractory B-cell non-Hodgkin lymphoma. Br J Haematol. 2007;138:502-505.
80. Khan KD, Emmanouilides C, Benson DM Jr, et al. A phase 2 study of rituximab in combination with recombinant interleukin-2 for rituximab-refractory indolent non-Hodgkin’s lymphoma. Clin Cancer Res. 2006;12:7046-7053.
81. Yang ZZ, Grote DM, Ziesmer SC, et al. Soluble IL-2Ralpha facilitates IL-2-mediated immune responses and predicts reduced survival in follicular B-cell non-Hodgkin lymphoma. Blood. 2011;118:2809-2820.
82. Niitsu N, Iijima K, Chizuka A. A high serum-soluble interleukin-2 receptor level is associated with a poor outcome of aggressive non-Hodgkin’s lymphoma. Eur J Haematol. 2001;66:24-30.
83. Morito T, Fujihara M, Asaoku H, et al. Serum soluble interleukin-2 receptor level and immunophenotype are prognostic factors for patients with diffuse large B-cell lymphoma. Cancer Sci. 2009;100:1255-1260.
84. Fabre-Guillevin E, Tabrizi R, Coulon V, et al. Aggressive non-Hodgkin’s lymphoma: concomitant evaluation of interleukin-2, soluble interleukin-2 receptor, interleukin-4, interleukin-6, interleukin-10 and correlation with outcome. Leuk Lymphoma. 2006;47:603-611.
85. Del Vecchio M, Bajetta E, Canova S, et al. Interleukin-12: biological properties and clinical application. Clin Cancer Res. 2007;13:4677-4685.
86. Ansell SM, Geyer SM, Maurer MJ, et al. Randomized phase II study of interleukin-12 in combination with rituximab in previously treated non-Hodgkin’s lymphoma patients. Clin Cancer Res. 2006;12:6056-6063.
87. Labidi SI, Menetrier-Caux C, Chabaud S, et al. Serum cytokines in follicular lymphoma. Correlation of TGF-beta and VEGF with survival. Ann Hematol. 2010;89:25-33.
88. Salles G, Bienvenu J, Bastion Y, et al. Elevated circulating levels of TNFalpha and its p55 soluble receptor are associated with an adverse prognosis in lymphoma patients. Br J Haematol. 1996;93:352-359.
89. Ho CL, Sheu LF, Li CY. Immunohistochemical expression of angiogenic cytokines and their receptors in reactive benign lymph nodes and non-Hodgkin lymphoma. Ann Diagn Pathol. 2003;7:1-8.