Effect of interleukin 21 and its receptor on CD8+ T cells in the pathogenesis of diffuse large B-cell lymphoma

Interleukin 21 (IL-21) and its receptor, IL-21R, play a key role in innate and adaptive immunity. In the present study, the effect of IL-21 and IL-21R on the pathogenesis of diffuse large B-cell lymphoma (DLBCL) was investigated. The serum levels of IL-21 were detected by enzyme-linked immunosorbent assay, and the expression of IL-21R on CD8+ T cells was examined through flow cytometry. The data showed that the serum level of IL-21 was significantly decreased in the patients with DLBCL compared with the healthy controls (P<0.001), whereas the expression of IL-21R was clearly elevated on the CD8+ T cells in the patients with DLBCL. Further analyses revealed that the downregulation of the IL-21 serum level was correlated with an increased tumor stage of DLBCL, while the expression of IL-21R on the CD8+ T cells was positively correlated with the tumor stage. Also, the serum level of IL-21 and the proportion of IL-21R on the CD8+ T cells were negatively correlated in the patients. Notably, it was identified that the proportion of IL-21R on the CD8+ T cells, but not the serum level of IL-21, was significantly upregulated in the patients with bone-marrow involvement and B symptoms. These results indicate that IL-21 and IL-21R may be involved in the pathogenesis of DLBCL, in which IL-21R may reflect the progression of the disease more accurately than the serum level of IL-21.


Introduction
Interleukin 21 (IL-21) belongs to the type I cytokine family and plays critical roles in the human immune system. IL-21 was originally believed to be produced by cluster of differentiation 4-positive (CD4 + ) T cells, among which, T-follicular helper cells could be a major source. Previous studies have shown that IL-21 is also produced by Th17 cells and by natural killer T cells, and that IL-21 mRNA can be detected in stromal cells in the lymph nodes. All these studies indicate the significance of IL-21 in innate and adaptive immune responses (1)(2)(3).
The IL-21 receptor (IL-21R) contains six tyrosines in the human cytoplasmic domains, Y281, Y361, Y369, Y397, Y317 and Y510 (4,5). Previous studies have reported that IL-21R and the common γ-chain (γc; CD132) function together as a heterodimer for IL-21 (6,7). Upon binding to the IL-21R expressed on cells lacking the γc, IL-21 is unable to transduce any intracytoplasmic signals, whereas in γc-transfected cells, IL-21 binds to the IL-21R and then activates signals downstream (7). Furthermore, a study on chemical cross-linking has previously demonstrated the direct binding of IL-21 to the γc (7). The expression of the IL-21R complex can be detected in various lymphoid organs, including the spleen and thymus. IL-21R is also expressed on T cells, B cells, dendritic cells, natural killer cells, keratinocytes and macrophages. A further study revealed that the functional IL-21R is expressed on CD4 + T cells and CD8 + T cells, and that this expression can be upregulated by the activation of the T cell receptor (3).
IL-21 plays significant roles in antitumor activity through the regulation of CD8 + T cells. It has been shown that IL-21 promotes CD8 + T-cell dependent tumor responses against solid tumors in a mouse model (8,9). IL-21 therapy in mice increases the number of tumor-infiltrating CD8 + T cells, expands the number of tumor-specific CD8 + T cells and protects the IL-21-treated mice against a recurrence of the same tumor (9). IL-21 treatment in humans causes the increased expression of perforin and granzyme B in

Effect of interleukin 21 and its receptor on CD8 + T cells in the pathogenesis of diffuse large B-cell lymphoma
CD8 + T cells, which induce apoptosis of B-cell chronic lymphocytic leukemia cells (10,11). These data indicate that CD8 + T cells may be incorporated into the IL-21-based therapies of hematological cancers. In the present study, the serum level of IL-21 and the expression of IL-21 on IL-21R with the pathogenesis of diffuse large B-cell lymphoma (DLBCL) was investigated.

Results
Clinical characteristics of the study subjects. Selected characteristics of the 72 patients with DLBCL and 62 controls are presented in Table I. The cases and controls did not reveal any statistical significance with regard to age (P>0.05) and gender (P>0.05). Of the 72 patients, eight were in Ann Arbor stage I, 10 were in stage II, 25 were in stage III and 29 were in stage IV. A total of 18 cases exhibited bone marrow involvement and 30 patients presented with B symptoms.

Serum level of IL-21 and expression of IL-21R on CD8 + T cells in cases and controls.
The serum levels of IL-21 were investigated in the 72 cases and 62 controls. As shown in Fig. 1A Fig. 1B). These data indicate a potential involvement of IL-21 and IL-21R in the pathogenesis of DLBCL.
Serum level of IL-21 and expression of IL-21R on CD8 + T cells in patients with various tumor stages. The Ann Arbor staging system is a standardized way for a cancer care team to summarize information with regard to how far a cancer has spread (11). Stage I is indicative of a cancer that is located in a single region, typically one lymph node and the surrounding area, whereas stage IV is indicative of the diffuse or disseminated involvement of one or more extralymphatic organs. The serum level of IL-21 and the expression of IL-21R on the CD8 + T cells were analyzed in the patients with various stages (Fig. 2). The serum level of IL-21 was significantly lower in the patients with stage III and IV compared with those with stages I and II ( Fig. 2A). The analysis of IL-21R demonstrated that the expression of IL-21R on the CD8 + T cells was clearly elevated with increasing stage (Fig. 2B). These results indicate that IL-21 and IL-21R could be associated with the progression of DLBCL.

Correlation between serum level of IL-21 and expression of IL-21R on CD8 + T cells in DLBCL.
Since a decreased level of serum IL-21, but increased expression of IL-21R on CD8 + T cells, was observed in the DLBCL samples, and due to the diverse correlations with disease progression, we hypothesized that a potential correlation existed between the serum level of IL-21 and the expression of IL-21R on the CD8 + T cells in the patients with DLBCL. The results data showed that the decrease in the serum level of IL-21 was significantly correlated with the increase in the expression of IL-21R on the CD8 + T cells in the patients with DLBCL (P<0.001; Fig. 3). With regard to bone marrow involvement, the serum level of IL-21 did not reveal a difference between the two groups ( Fig. 4A), while the expression of IL-21R on the CD8 + T cells was higher in the positive group (31.1±2.1 vs. 25.4±1.2%, P=0.020; Fig. 4B). Similarly, the patients with or without B symptoms did not present different serum levels of IL-21, but showed a higher expression of IL-21R when B symptoms were present (Fig. 5). These data indicated that the expression of IL-21R on CD8 + T cells may be used as a prognostic marker for DLBCL.

Discussion
In the present study, IL-21 and its receptor were analyzed in CD8 + T cells, and it was identified that IL-21/IL-21R are closely involved in the development and progression of DLBCL. Several studies conducted on murine models have confirmed the antitumor role of IL-21 (12)(13)(14). DLBCL is a heterogeneous disease with multiple subtypes and varying clinical outcomes. It has been reported that IL-21R expression is positive on primary CD10 + DLBCL cells and DLBCL cell lines (15)(16)(17). IL-21 is known to induce apoptosis in de novo DLBCL primary tumors, however, it may not effect the viability of human healthy B cells (15). Additionally, IL-21 is able to induce tumor regression and increase the survival of mice with xenograft DLBCL tumors (17). IL-21 stimulates the apoptosis of the DLBCL cell line, CRL-2632, by activating JAK1, JAK3, STAT1 and STAT3 (15). In addition, it appears that the anti-lymphoma effects of IL-21 are dependent on a mechanism involving the IL-21-activated STAT3 upregulation of c-Myc, in which c-Myc is a highly prognostic marker in DLBCL (17,18). The present study confirmed the critical role of IL-21 in the development of DLBCL by identifying a decreased serum level of IL-21 in the patient group (Fig. 1A). A negative correlation was also found between the serum level of IL-21 and the tumor stage, indicating the involvement of this cytokine in the progression of the disease (Fig. 2A).
The effects of IL-21 on CD8 + T cells are varied. IL-21 increases the proliferation of murine or human mature T cells stimulated with anti-CD3 or its antigen (19). In conjunction with T-cell receptor stimulation and other common γc cytokines, including IL-7 and IL-15, IL-21 is able to augment the proliferation and differentiation of human and mouse CD8 + T cells into potent cytolytic effectors (19,20). In the present study, an elevated level of IL-21R was observed on the peripheral CD8 + T cells in the DLBCL cases (Fig. 2B), and this positively correlated with the tumor grade. Notably, the level of IL-21R on the peripheral CD8 + T cells, but not the serum level of IL-21, was associated with bone marrow involvement and B symptoms, indicating that IL-21R on peripheral CD8 + T cells may play more significant roles in the progression of DLBCL.
In conclusion, the present study identified a decreased level of serum IL-21, but an increased level of IL-21R expression on the CD8 + T cells in the patients with DLBCL. Additionally, the analyses revealed opposite correlations between IL-21 and IL-21R with disease progression, in which IL-21R on CD8 + T cells may further reflect the prognosis of the disease. These data shed light on understanding the pathogenesis of DLBCL and provide knowledge for the use of IL-21 as a novel therapy.