Virchows Archiv

, Volume 456, Issue 6, pp 615–623

Morphometric analysis of the balance between CXCR3+ T cells and FOXP3+ regulatory T cells in lymphocyte-rich and conventional gastric cancers

Authors

    • Department of Pathology, Mito Medical CenterNational Hospital Organization
  • Osamu Yoshie
    • Department of MicrobiologyKinki University School of Medicine
Original Article

DOI: 10.1007/s00428-010-0921-9

Cite this article as:
Ohtani, H. & Yoshie, O. Virchows Arch (2010) 456: 615. doi:10.1007/s00428-010-0921-9

Abstract

Lymphocyte-rich gastric carcinomas (Ly-rich GCs) are characterized by the formation of lymphoid stroma. Our previous study has shown abundant infiltration of CXCR3+ T cells and their frequent clustering with CXCL9 (monokine induced by interferon-γ)-expressing stromal cells in the lymphoid stroma of Ly-rich GCs. Foxp3+ regulatory T cells (Tregs) suppress immune responses in the peripheral tissues and play a role in the immunosuppression of cancer tissues. The present study was therefore undertaken to evaluate the significance of the balance between CXCR3+ T cells and Tregs in 44 Ly-rich and 37 conventional GCs by morphometrical analyses of immunohistochemistry. Compared with the pronounced infiltration of CXCR3+ T cells in the lymphoid stroma, the numbers of Foxp3+ cells were relatively low in Ly-rich GCs. Therefore, the ratios of CXCR3+/Foxp3+ cells were much higher in Ly-rich GCs than in conventional GCs. This suggests the occurrence of T-helper type 1 (Th1)-shifted immune responses in Ly-rich GCs. On the other hand, conventional GCs were characterized by a paucity of CXCR3+ T cells and a relative abundance of Tregs. Furthermore, the stroma inside the cancer was characterized by even less CXCR3+ cells, suggesting a strongly immunosuppressive microenvironment. Since Tregs are known to express CCR4, we also examined the tissue distribution of cells expressing its ligand CCL22. CCL22 was not detected in conventional GCs and only sparsely detected in dendritic cells but not in cancer cells in Ly-rich GCs. To conclude, Tregs may play a more important role in conventional GCs than in Ly-rich GCs from the viewpoint of immunosuppression.

Keywords

Gastric cancerLymphoid stromaCXCR3Regulatory T cellsTumor immunity

Introduction

Human cancer tissue is associated with varying degrees of tumor-infiltrating lymphocytes (TILs). Although the significance of TILs as an indicator of host immune responses against cancer cells may not be so straightforward [1], TILs or a subset of TILs are generally associated with a better prognosis of patients in various cancers, suggesting their protective role in antitumor immunity [26]. As an extreme variation, some cancers are characterized by a prominent lymphocytic reaction in the stroma. Among gastric cancers (GCs), such cases are designated as “gastric cancer with lymphoid stroma” or “lymphoepithelioma-like carcinoma (LELC).” They generally show a better prognosis than conventional GCs [7, 8]. Cancer cells in typical LELC are frequently associated with Epstein-Barr (EB) virus infection [9, 10]. In the present paper, we use the term “lymphocyte-rich GCs (Ly-rich GCs)” to include typical LELC and other GCs associated with a rich distribution of lymphocytes in the stroma (for details, see the first section of “Materials and methods”).

Type 1 immune responses are exerted by type 1 effector T cells (T-helper type 1 [Th1] cells, cytotoxic T cells [CTLs]) and natural killer cells (NK cells) and are important in host defense against intracellular pathogens and tumor cells. CXCR3 is one of the representative chemokine receptors for type 1 immune cells, especially Th1 cells [11, 12]. Previously, we have shown that the majority of TILs in lymphocyte-rich GCs express CXCR3, while CXCL9 (also called monokine induced by interferon-γ [MIG]), one of the cognate chemokine ligands of CXCR3, is abundantly expressed by dendritic-shaped stromal cells [13]. As a result, frequent “CXCR3+ T cell–CXCL9+ stromal cell clustering” was observed, which featured the lymphoid stroma of Ly-rich GCs [13]. Furthermore, CXCR3+ T cells clustering with CXCL9+ stromal cells are frequently positive for Ki-67, suggesting some ongoing immune responses [13].

Recent advances in tumor immunology have revealed that cancers develop by escaping the host immune responses through stepwise processes [14, 15]. Consequently, established cancer tissue is generally considered to be a strongly immunosuppressive microenvironment. In this context, our above-mentioned results from Ly-rich GCs may seem rather inconsistent to the current concept. Therefore, to understand the immunosuppressive aspect of GCs, the present study was undertaken to analyze the potential role of forkhead box protein-3 (Foxp3)-positive regulatory T cells (Tregs) as the mediator of immunosuppression [16, 17]. We compared the distribution density of CXCR3+ cells and that of Foxp3+ cells by morphometric analyses. We reveal here that Ly-rich GCs are characterized not only by the abundance of CXCR3+ cells but also a relative paucity of Foxp3+ cells, resulting in a high ratio of CXCR3+ cells to Foxp3+ cells (termed here as “CXCR3/Foxp3 ratio”). On the other hand, conventional GCs (i.e., GCs other than Ly-rich GCs) are characterized by a relative abundance of Foxp3+ cells and a low CXCR3/Foxp3 ratio, especially within the cancer stroma. Thus, our findings may provide a new insight into the possible differences in immunosuppressive mechanisms between Ly-rich GCs and conventional GCs.

Materials and methods

Materials

We use the term “lymphocyte-rich gastric cancer (Ly-rich GC)” to include both LELC and other GCs associated with abundant TILs in the whole stroma (see [13] for details). The present study enrolled 44 cases of Ly-rich GCs (EBV+: 35 cases; EBV: 9 cases) and 37 cases of conventional GCs (EBV+: 2 cases; EBV: 35 cases). The 35 EBV cases of conventional GCs were consecutively sampled by their histopathological numbering. Intramucosal carcinoma (pTis and pT1[M]) were not included in this study, because typical lymphocyte-rich stroma is observed only when carcinoma cells have started invading the submucosa. All cases were staged as described by tumor, node, and metastasis (TNM) Classification, 7th edition [18]. The present study was approved by the ethical committee of Mito Medical Center.

Immunohistochemistry (single-labeling)

Enzyme-linked immunohistochemistry (immunoperoxidase method) was performed using formalin-fixed, paraffin-embedded tissue sections as described previously [13]. The EBV status was checked by in situ hybridization for EBER in all cases [13]. The primary antibodies used in this study were mouse monoclonal antibodies to human CXCR3 (CD183; clone 1C6, IgG1; BD, Franklin Lakes, NJ; used at 1:400 = 1.25 μg/mL) and Foxp3 (clone 236A/E7, IgG1; Abcam, Cambridge, MA; used at 1:100), and affinity-purified rabbit polyclonal antibody to human CCL22 (MDC) (PeproTech, Rocky Hill, NJ; used at 1:250 = 4 μg/mL). Tissue sections were incubated with primary antibodies overnight. The staining methods of other lymphocyte markers were described previously [13]. Heat antigen retrieval methods applied before incubation with primary antibodies were as follows: for anti-CXCR3 and anti-CCL22, 10 mmol/L Tris HCl buffer + 1 mmol/L EDTA, pH 9.0, at 95°C for 60 min; for anti-Foxp3, 1:5 dilution of DAKO (Glostrup, Denmark) high-pH buffer (S3308, DAKO), at 95°C for 60 min. Anti-mouse or anti-rabbit Envision (DAKO) was used as the secondary antibody. Diaminobenzidine (DAB; DAKO) was used as the chromogen. Endogenous peroxidase activity was inactivated by immersing tissue sections in 3% H2O2 for 5 min after incubation with primary antibodies.

Enzyme-linked double immunohistochemistry for CD4 or CD8 and Foxp3

The immunoperoxidase method for CD4 or CD8 detection was also performed as described above. Tissue sections were then treated with a 1:5 dilution of DAKO high-pH retrieval solution at 95°C for 20 min to inactivate antibodies and enzymes used in the first step. Then immunohistochemistry for Foxp3 was performed using a 1:40 dilution of anti-Foxp3 antibody. TrueBlue (KPL, Gaithersburg, MA) was used as the chromogen.

Morphometry

The percentage of Foxp3+ cells among CD4+ or CD8+ cells (i.e., the labeling index of Foxp3) was determined using tissue sections double-stained for CD4 or CD8 and Foxp3. The number of total CD4+ (or CD8+) cells and that of CD4+ (or CD8+) Foxp3+ was manually counted in the same areas using an ocular grid (10 × 10-mm lattice) with a 400× microscopic field (40× objective lens and 10× ocular lens using a BX51 microscope; Olympus, Tokyo, Japan). The area of one lattice was 0.0625 mm2. At least three fields were counted for each case, and the numbers were averaged. The labeling index of Foxp3+ cells among CD4+ (or CD8+) cells was calculated by the following formula: the number of CD4+ (or CD8+) Foxp3+ cells divided by the total number of CD4+ (or CD8+) cells in the same field. This analysis was performed in five representative cases of Ly-rich GCs. We also measured the distribution density of CXCR3+ cells and that of Foxp3+ cells in all 81 cases of GC by the same method described above. The results were expressed by the average number of three areas. In this analysis, the most densely distributed areas were selected for cell counting. The size of cancer tissues we examined were 2 to 4 cm in length.

All the statistical analyses were performed using PASW Statistics 18 (SPSS Inc., Chicago, IL), including the Fisher's exact test for the test of contingency table, the Kruskal-Wallis test for the comparison between unpaired two groups, the Pearson correlation coefficient between two variables, and the discriminant analysis to check how variables discriminate two groups. All the tests were judged to be significant when p < 0.05.

Double-labeling immunofluorescence method for CCL22 and CD83

Tissue sections were first immersed in a 1:5 dilution of high-pH antigen retrieval solution at 95°C for 60 min. Nonspecific binding was blocked by Protein Block (X0909, DAKO). The sections were then incubated with a mixture of rabbit antihuman CCL22 (1:100) and mouse monoclonal antihuman CD83 (1:30; Clone 1H4b; Novocastra, Benton Lane, UK) overnight. Alexa fluor 488-labeled donkey antirabbit IgG (1:100 = 20 μg/mL; Molecular Probe, Carlsbad, CA) and Alexa fluor 555-labeled donkey antimouse IgG (1:100 = 20 μg/mL) were applied in a mixture for 30 min. After DAPI (Molecular Probes) nuclear staining, specimens were mounted with ProLong Gold (Molecular Probes). Immunofluorescent observation was performed with a confocal laser scanning microscope (TCS SP5; Leica Microsystems, Wetzlar, Germany).

Results

General histopathological aspects

Forty-four cases of Ly-rich GCs were composed of 25 typical LELCs (EBV+, 22 cases; EBV, 3 cases) and 19 other Ly-rich GCs (EBV+, 13 cases; EBV, 6 cases). LELCs were characterized by poor differentiated carcinoma cells with a syncytial appearance and abundant TILs in the whole stroma [13] (Fig. 1a, b). Of 37 cases of conventional GCs, 35 were negative for EBER. Table 1 shows the stage distribution of all the cases. There was a significant difference in the stage distribution between Ly-rich GCs and conventional GCs (p < 0.0005, Fisher’s exact test); i.e., the Ly-rich GCs were composed of cases with less advanced stages than the conventional GCs (Table 1A). Similarly, typical LELCs showed less advanced stages than other GCs (Table 1B). There was no significant correlation between the numbers of CXCR3+ cells and stages or between the numbers of Foxp3+ cells and stages in either Ly-rich or conventional GCs (supplementary figures).
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Fig. 1

a, b Hematoxylin and eosin staining of Ly-rich GC. The Ly-rich GCs are characterized by an abundance of TILs and a pushing margin. In typical LELCs, cancer cells show poorly differentiated morphology as in this case. c, d Immunohistochemistry for CXCR3 (c) and Foxp3 (d) (performed in all cases). Note the overall abundance of CXCR3+ cells over Foxp3+ cells. e, f Double immunohistochemistry for CD4 and Foxp3 (e) and CD8 and Foxp3 (f) clearly demonstrates that nearly all Foxp3+ are CD4+ (performed in five representative cases of Ly-rich GCs). Scale bars: 100 μm (a, c, d), 5 μm (b), and 20 μm (e, f)

Table 1

Stage distribution of cases

 

I

II

III

IV

Total

A. Ly-rich vs. conventional GCs

 Ly-rich GCs (%)

21

17

5

1

44

 

(47.7)

(38.6)

(11.4)

(2.3)

 

 Conventional GCs (%)

10

12

10

5

37

 

(27.0)

(32.4)

(27.0)

(13.5)

 

B. LELC (a subgroup of Ly-rich GC) vs. others

 LELC (%)

16

7

2

0

25

 

(64)

(28)

(8)

(0)

 

 Others (%)

15

22

13

6

56

 

(26.8)

(39.3)

(23.2)

(10.7)

 

p < 0.0005 (Fisher’s exact test)

The distribution of stage is different from our previous paper [13] by adopting the 7th ed. of TNM classification [18]

Nearly all Foxp3+ cells are CD4+ cells in Ly-rich GCs

In Ly-rich GCs, Foxp3+ cells were distributed relatively evenly in the whole area of the stroma. They constituted only a small part of the TILs as compared with abundantly distributed CXCR3+ cells (Fig. 1c, d). Previously, we have shown that the average frequencies of Foxp3+ cells among CXCR3+ cells and CCR4+ cells were 5.1% and 47%, respectively, in Ly-rich GCs [13]. To further characterize Foxp3+ cells, we counted the Foxp3-labeling index among CD4+ and CD8+ cells by double staining in five representative Ly-rich GC cases (Fig. 1e, f). The Foxp3-labeling index among CD4+ cells and CD8+ cells were 98% and 2%, respectively. Therefore, we judged that Foxp3+ cells in Ly-rich GC were mostly CD4+ regulatory T cells (Tregs). This conclusion is consistent with a recent report [19].

Ly-rich GCs are characterized by a relative paucity in Tregs

We next counted the numbers of CXCR3+ cells and Foxp3+ cells in all GC cases. Note that we selected areas rich with TILs as described in “Materials and methods.” As shown in Fig. 2a, the numbers of CXCR3+ cells in Ly-rich GCs were approximately three-fold higher than those in conventional GCs, as expected from our previous study [13]. In contrast, the numbers of Foxp3+ cells in Ly-rich GCs showed an approximately 1.5-fold increase (Fig. 2b). Accordingly, the CXCR3/Foxp3 ratio becomes higher in Ly-rich GCs than in conventional GCs (Fig. 2c).
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Fig. 2

a–c Box-whisker plots of the number of CXCR3+ cells (a), the number of Foxp3+ cells (b), and CXCR3/Foxp3 ratio (c) between Ly-rich- and conventional GCs (including all cases). All variables in (a–c) showed a statistically significant difference (Kruskal-Wallis test). d A scatter diagram showing the number of CXCR3+ cells and the CXCR3/Treg ratio (open circles, Ly-rich GCs; closed triangles, conventional GCs; including all cases). The number of CXCR3+ cells correlates with the CXCR3/FoxP3 ratio in total GCs. Also note that the Ly-rich and conventional GCs are well discriminated by these two parameters. Vertical lines in a, b, and d show the number of cells in one unit area (one lattice in a 400× field, 0.0625 mm2)

We next analyzed the relationship between various parameters. When all GCs were included, the number of CXCR3+ cells positively correlated with the CXCR3/Foxp3 ratio (γ = 0.55, p < 0.0005 [Pearson]; Fig. 2d) and also correlated with the number of Foxp3+ cells (γ = 0.53, p < 0.0005 [Pearson], not shown). Discriminant analysis revealed that the three variables (CXCR3+ cells, Foxp3+ cells, and CXCR3/Foxp3 ratio) contributed to 92.6% discrimination between Ly-rich GCs and conventional GCs. The analyses here also indicated that although the numbers of CXCR3+ and Foxp3+ cells as a whole were in good correlation, the more CXCR3+ T cells infiltrated the cancer tissue, the more CXCR3+ T cells predominated over Tregs

Uneven distribution of TILs in conventional GCs

The results in the previous section also revealed that conventional GCs were characterized by a low CXCR3+/Treg ratio. It should be remembered that the above morphometry was performed using areas with relatively abundant TILs. As we previously reported for colorectal cancer [20], TILs in conventional GCs also tended to be abundant along the invasive margin (cancer–host interface; Fig. 3a). The stroma inside the cancer, i.e., the cancer stroma excluding areas along the invasive margin, was usually sparse in TILs (Fig. 3b). We therefore next focused our attention on the distribution of Tregs and CXCR3+ cells between these two areas. For this purpose, we selected 12 cases of conventional GCs, in which the invasive margin only contained a substantial number of TILs. The remaining 22 cases of conventional GCs showed either a mild increase of TILs along the invasive margin or overall scantiness of TILs. The histologic types of the selected cases were tubular and/or papillary adenocarcinoma or poorly differentiated adenocarcinoma of solid type (“por1” by Japanese classification). No scirrhous carcinomas (diffuse type by Lauren's classification [21]; “por2” or “sig” by Japanese classification [22]) were included in these 12 cases. Figure 3c shows a typical scirrhous carcinoma (“por2”), where TILs were only infrequently observed.
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Fig. 3

a, b Representative hematoxylin and eosin staining of the invasive margin (a) and the stroma inside the cancer (b) in a conventional GC (tubular adenocarcinoma), showing that TILs are more abundant along the invasive margin. Arrows in a indicate the invasive margin. c Diffuse type GCs are usually poor in TILs. Scale bars: 50 μm (a–c)

Stroma inside the cancer in conventional GCs is characterized by a paucity of TILs and a decrease of CXCR3/Treg ratio as compared with the invasive margin

The morphometrical analyses were done in the abovementioned 12 cases of conventional GCs. As shown in Fig. 4, the stroma inside the cancer showed less CXCR3+ cells and much lower CXCR3/Treg ratios than the invasive margin. Discriminant analysis revealed that the number of CXCR3+ cells and the CXCR3/Foxp3 ratio contributed to 100% discrimination between the invasive margin and the stroma inside the cancer. The data here indicate that the stroma inside the cancer of conventional GCs is characterized by relative abundance of Tregs over effector CXCR3+ cells.
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Fig. 4

Scatter diagram showing the number of CXCR3 and CXCR3/Treg ratio in 12 cases of conventional GCs. This shows that both the number of CXCR3 and the CXCR3/FoxP3 ratio were lower in the stroma inside the cancer than in the invasive margin. Vertical line: the number of CXCR3+ cells in one unit area (one lattice in a 400× field, 0.0625 mm2)

Sporadic dendritic cells (DCs) express CCL22

Tregs are known to frequently express CCR4, and the chemokine CCL22 is reported to be involved in the Treg infiltration of ovarian tumors [23]. We therefore analyzed CCL22 expression in five representative cases of Ly-rich GCs and six cases of conventional GCs. As shown in Fig. 5a, CCL22 was only sparsely expressed by stromal cells in four of five Ly-rich GCs, while no such cells were detected in six conventional GCs. Cancer cells were totally negative for CCL22 in all these cases. Although the distribution density of CCL22+ cells was less than that of CD83+ mature DCs in Ly-rich GCs (Fig. 5b), most CCL22+ cells coexpressed CD83 and showed dendritic morphology together with close cell-to-cell contacts with neighboring lymphocytes, as confirmed in two representative Ly-rich GCs (Fig. 5c). The data here indicated that a small portion of mature DCs but not cancer cells expressed CCL22 in Ly-rich GCs.
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Fig. 5

a, b Immunohistochemistry for CCL22 (MDC) (a) and CD83 (b) in Ly-rich GCs (performed in five cases of Ly-rich GCs and six cases of conventional GCs). Note only a sporadic distribution of CCL22+ cells in the stroma (inset, oil-immersion lens) as compared with mature DCs detected by CD83. c Confocal laser scanning microscopy revealed that CCL22+ cells coexpress CD83+, a mature DC marker (performed in two cases of Ly-rich GCs). Note the close cell-to-cell contacts of CCL22+ DCs with cytoplasmic processes, labeled in red, with neighboring lymphocytes. Scale bars: 50 μm (a, b) and 10 μm (c)

Discussion

The present study has shown that the lymphoid stroma of Ly-rich GCs is characterized not only by a drastic increase in CXCR3+ T cells but also by a relative paucity of Tregs, suggesting a microenvironment highly enriched with Th1 cells. In sharp contrast, conventional GCs are characterized by a low number of CXCR3+ T cells and a relative increase in Tregs, suggesting a highly immunosuppressive milieu. These results may raise an interesting issue in terms of tumor immunity and immune evasion in these two groups of GC.

Our previous report was mainly focused on Th1/Th2 responses in Ly-rich GCs and demonstrated a predominant infiltration of CXCR3+ cells over CCR4+ cells [13]. Tregs suppress the type 1 and type 2 immune responses and are regarded to be important for the control of immune responses [16, 17]. Tregs are also considered to participate in the immune evasion mechanism of cancer [23]. Therefore, the effector cell–Treg balance could be important for understanding the immune status of cancer tissues. In fact, a high CD8 to Treg ratio in cancer tissues was associated with a prolonged survival of patients with cancer of the ovary [4], liver [24], and kidney [25]. We therefore focused our attention on the balance between CXCR3+ T cells and Tregs in Ly-rich and conventional GCs. By analyzing the CXCR3/Foxp3 ratio, our present study has shown that Ly-rich GCs are characterized by a relative paucity of Tregs in the abundance of CXCR3+ TILs. This, in turn, underscores a relative abundance of Tregs along with a general paucity of TILs in conventional GCs. Therefore, Tregs may play a less major role in Ly-rich GCs than in conventional GCs as the mediator of immune evasion. The immune cell infiltrate in conventional GCs, if present, also tends to be more prominent along the invasive margin than in the stroma inside the cancer. The analysis of the 12 selected cases of conventional GCs clearly demonstrated that the stroma within the cancer is associated with even less CXCR3+ T cells (i.e., less TILs) and more Tregs than the invasive margin. The results from conventional GCs are thus fully consistent with the current concept that tumor tissue is a strongly immunosuppressive microenvironment. We may regard the findings from conventional GCs as the basic feature of cancer tissue.

Concerning the function of Tregs in conventional GCs, Mizukami et al. [26] have reported that cases with diffuse distribution of Tregs are associated with a poorer prognosis than cases with peritumoral distribution of Tregs. However, Ly-rich GCs, usually associated with a better prognosis, generally show diffuse distribution of Tregs as shown in the present paper. Therefore, the balance between effector T cells and Tregs may also have a strong impact on the significance of TILs.

Tregs are known to frequently express CCR4, and Curiel et al. [23] have reported that cancer cells and tumor-associated macrophages produce CCL22 to attract CCR4+ Tregs to ovarian cancer tissues. For GCs, Mizukami et al. [27] have reported that CD14+ cells in the tumor stroma express CCL17 or CCL22. Our present data showed that CCL22 is only sporadically expressed by a part of CD83+-mature DCs but not by cancer cells in Ly-rich GCs. The production of CCL22 by cancer cells may be dependent on the type of cancer. DC expression of CCL22 is consistent with a previous report [28]. As shown in “Results,” the absolute number of Tregs is higher in Ly-rich GCs than in conventional GCs (Fig. 2b), with the number of Tregs being approximately 10% to 20% of CXCR3+ T cells in Ly-rich GCs (Fig. 2d). Thus, the expression of CCL22 by a part of DCs could, at least partly, explain the increased recruitment of Tregs in Ly-rich GCs. We may need further analyses to clarify the mechanism(s) of Treg recruitment in Ly-rich GCs and conventional GCs.

Given that Tregs may not be a dominant immunosuppressive factor in Ly-rich GCs, we may need to analyze possible roles of other immunosuppressive factors such as immunosuppressive cytokines TGF-β and IL-10 in the immune evasion mechanism of Ly-rich GCs. In particular, Ly-rich GCs are frequently associated with EBV, which is known to induce IL-10 [29]. Another important issue to be addressed in the future is the possible involvement of IL-17-producing T helper (Th17) cells in Ly-rich GCs, which are known to play the major role in chronic inflammation and autoimmune pathogenesis, and selectively express CCR6 [30]. These analyses are now ongoing.

In conclusion, our present morphometrical analyses on the balance between CXCR3+ T cells and Foxp3+ Tregs have revealed that Ly-rich GCs are characterized by an absolute and relative abundance of CXCR3+ effector T cells over Tregs in contrast to conventional GCs. On the other hand, conventional GCs, particularly in the stroma inside the cancer, are characterized by an absolute paucity of CXCR3+ effector T cells and a relative abundance of Tregs. The data suggest that Tregs may play a more dominant role in conventional GCs than in Ly-rich GCs from the viewpoint of immunosuppression in cancer tissue. The present findings also highlight the unique immunologic aspect of Ly-rich GCs and the complexity of immune evasion mechanisms used by these types of cancer.

Acknowledgments

We are grateful to Dr. Masaaki Miyazawa (Kinki University School of Medicine) for critical reading of the manuscript, Dr. Takashi Nakayama (Kinki University School of Medicine) for valuable discussion, Dr. Noriko Kimura (Hakodate National Hospital) and Dr. Hiroshi Naganuma (Sendai City Hospital) for supplying surgical materials, Ms. Fumiko Date (Tohoku University Graduate School of Medicine) for clerical assistance, and Ms. Megumi Maeda and Ms. Shoko Kajiwara (National Institute for Materials Science [NIM.S.]–Leica Bioimaging Laboratory) for technical assistance in confocal laser scanning microscopy. The present study was partly supported by the National Hospital Organization Collaborative Clinical Research Grant as well as the “Nanotechnology Network Project” and “HAITEKU” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

428_2010_921_Fig1_ESM.jpg (205 kb)
Supplementary figures

The number of CXCR3+ cells and that of Foxp3+ cells show no significance changes with stages in both Ly-rich and conventional GCs (JPEG 205 kb)

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