Purpose: The role of infiltrating B cells in hepatocellular carcinoma has been overlooked for many years. This study is aimed to delineate the distribution, prognostic value, and functional status of B cells in human hepatocellular carcinoma.

Experimental design: Immunohistochemistry was used to investigate the distribution and clinical significance of infiltrating CD20+ B cells in a series of 120 patients with hepatocellular carcinoma. The results were further tested in an independent series of 200 patients with hepatocellular carcinoma. The functional status of CD20+ B cells was determined by flow cytometry, immunofluorescence, and in vitro coculture assay.

Results: Infiltrating CD20+ B cells were predominantly concentrated in the tumor invasive margin, compared with the peri- and intratumor areas. High density of margin-infiltrating B lymphocytes (MIL-B) positively correlated with small tumor size, absence of vascular invasion, and increased density of CD8+ T cells (P < 0.05). Survival analyses revealed that increased number of MIL-Bs and their penetration through the tumor capsule were significantly associated with improved overall and recurrence-free survival, and were identified as independent prognosticators for patients with hepatocellular carcinoma (P < 0.05). Importantly, the results were further validated in another independent hepatocellular carcinoma cohort. Moreover, we found that MIL-Bs featured an atypical memory phenotype (IgDIgG+CD27CD38), expressed surface markers characteristic of antigen-presenting cells, possessed tumor-killing potential by producing IFN-γ, interleukin 12p40 (IL-12p40), granzyme B, and TRAIL, and acted in cooperation with CD8+ T cells.

Conclusions: The profile of CD20+ B cells in situ is a new predictor of prognosis for patients with hepatocellular carcinoma and provides a novel target for an optimal immunotherapy against this fatal malignancy. Clin Cancer Res; 19(21); 5994–6005. ©2013 AACR.

Translational Relevance

The presence and role of B cells in tumor immunity have been ignored for many years. Herein, we detected that the majority of infiltrating CD20+ B cells in hepatocellular carcinoma were enriched in tumor margin (MIL-Bs) and had an atypical memory phenotype (IgDIgG+CD27CD38). Moreover, we found and validated that the density of MIL-Bs and their penetration through tumor capsule were significantly and independently associated with prolonged survival and reduced recurrence in patients with hepatocellular carcinoma. Furthermore, our phenotypic and functional assays suggested that B cells were capable of facilitating the antitumor responses by serving as antigen-presenting cells, releasing immune-stimulating cytokines, and participating in the direct-killing effect through granzyme B and TRAIL. Thus, our results bring strong evidence that MIL-B is a powerful predictor for patient prognosis, and infiltrating B cells may be served as a potential target for immunotherapy in hepatocellular carcinoma.

Hepatocellular carcinoma is the second leading cause of cancer-related deaths worldwide, with an estimated 750,000 newly diagnosed cases annually. Liver transplantation and tumor resection have been proved to be the most effective standard therapies (1). However, the high rate of postsurgical recurrence (50–70% at 5 years) renders this disease a major challenge. Etiologically, chronic inflammation, mainly resulting from hepatitis virus infection, has long been accepted as a key contributor to hepatocarcinogenesis (2).

Recently, evidence has been accumulating that the inflammatory milieu, in particular the composition, localization, and nature of the infiltrating cells, may alter tumor biologic behavior and affect disease progression in hepatocellular carcinoma. In this regard, we and others have previously shown that dendritic cells, memory T cells, cytotoxic T cells, and γδT cells dwelling in the tumor bed tend to reduce hepatocellular carcinoma (HCC) growth (3–7), whereas M2 macrophages, N2 neutrophils, Th17 cells, and Foxp3+ regulatory T cells may stimulate HCC progression (8–11). These findings are in accordance with the general view that hepatocellular carcinoma is an immunogenic tumor, and provide a rationale for designing immunotherapies for HCC treatment. However, these studies mainly focused on the antitumor activities of T cells in cellular immune response. The analysis of the cellular component of humoral immunity to tumors might lead to the exploration of new targets for immunotherapy.

As the central component of humoral immunity, B lymphocytes function in antibody production, antigen presentation, and proinflammatory cytokines secretion. Initial studies in mouse tumor models suggested that B cells generally inhibited T-cell responses (12–14). More recently, the role of CD20+ (a B-lymphocyte marker expressed on mature B cells but not on plasma cells) lymphocytes in promoting favorable outcomes in several human cancers has been reported (15, 16), and the underlying mechanisms might include B cells producing tumor-specific antibodies, presenting tumor-specific antigen to T cells, and secreting cytokines that enhance antitumor immunity in the local tumor environment (17). In addition, the presence of infiltrating B lymphocytes in hepatocellular carcinoma has also been documented (18). However, the clinical relevance and prognostic significance of infiltrating B cells in hepatocellular carcinoma, as well as their microanatomical distribution and functional status, remain largely unknown.

In this study, we first delineated the microanatomical distribution of CD20+ B cells, and found most B cells were enriched in the tumor invasive margin, which we defined as margin-infiltrating B lymphocytes (MIL-B). The relationship between density of MIL-Bs and patient clinical outcome was investigated via immunohistochemistry of samples from 120 patients with hepatocellular carcinoma, and then tested in an independent series of 200 hepatocellular carcinoma cases. In addition, infiltrating B cells were isolated to investigate their phenotypes, functional status, and interactions with CD8+ T cells in vitro.

Patients and specimens

For the initial cohort, 120 patients with hepatocellular carcinoma were randomly selected from patients who had received curative hepatectomy between 2005 and 2007 in our institute. The median duration of follow-up was 61.5 months (range, 2.0–84.8 months). For validation, a total of 200 consecutive patients with hepatocellular carcinoma who had undergone curative hepatectomy between April and October 2006 were enrolled, and the median duration of follow-up was 59.5 months (range, 2.0–73.2 months). The inclusion and exclusion criteria of patients, postoperative surveillance, and treatment modalities have been described previously (19). Overall survival (OS) was defined as the time between the surgery and death and recurrence-free survival (RFS) was defined as the time between the surgery and recurrence. Patients without recurrence or death were censored at the last follow-up. There were no significant differences in the clinicopathologic features between the initial and the validation cohorts (Supplementary Table S1). Informed consent form was signed by each patient, and ethical approval was obtained from the Zhongshan Hospital Research Ethics Committee (Shanghai, PR China).

Immunohistochemistry and evaluation of immunohistochemical variables

Immunohistochemistry was conducted as described previously (20), using mouse anti-human CD20 (1:200 dilution, clone L26, Dako) and CD8 (1:50 dilution, clone 144B, Abcam) monoclonal antibodies (mAb) as the primary antibodies. Tumor sections were microanatomically divided into peritumor, invasive margin, and intratumor areas. The invasive margin was defined as the region within 500 μm on each side of the border between the tumor and normal liver tissue (21). The density of positive cells was evaluated as described previously (11). Briefly, five random microscopic fields (magnification, × 40) in each area were selected and captured. Positively stained cells were counted manually by two independent investigators blinded to the clinicopathologic data. The density of positive cells was calculated by averaging. Variations over a range of 5% were reevaluated for a consensus result.

Isolation of infiltrating lymphocytes

Fresh tumor specimens from 10 patients with hepatocellular carcinoma were obtained immediately after surgery. The specimens were also divided into peritumor, invasive margin, and intratumor areas as described above, by extending the margin area to 5 mm on each side of the border to obtain enough tissue. The tissues were minced and digested in 1 mg/mL collagenase (Invitrogen) and 50 UI/mL hyaluronidase (Sigma-Aldrich) for 1 hour. The cell suspension was filtered with a 400-mesh sieve and separated by centrifugation through a discontinuous Percoll (GE Healthcare) gradient with densities of 1.06 and 1.08 g/mL. The lymphocytes enriched at the interface of Percoll solutions were harvested and cultured in RPMI-1640 (Invitrogen) supplemented with 10% FBS (Invitrogen), 100 UI/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% humidified CO2 for further experiments.

Flow cytometric analysis

Flow cytometry was used to detect the surface marker and cytokine expression of CD20+ B cells isolated from the 10 patients with hepatocellular carcinoma. Peridinin chlorophyll (PerCP)-labeled anti-CD20, phycoerythrin (PE)-labeled antibodies to immunoglobulin D (IgD), IgM, IgG, interleukin 4 (IL-4), IL-6, IL-10, IFN-γ, and TRAIL; allophycocyanin (APC)-labeled anti-IL-2, IL-12p40, and CD38; and fluorescein isothiocyanate (FITC)-labeled antibodies to HLA-ABC, HLA-DR, CD40, CD80, CD86, CD27, CD8, and granzyme B were purchased from BioLegend. CD20+ B cells were sorted by flow cytometry from isolated lymphocytes and then stained with PerCP-labeled CD20 antibody. The viability of CD20+ B cells, detected by Trypan blue staining, was about 99% just after sorting, and over 85% after 3-day in vitro culture. For cytokine detection, CD20+ B cells were stimulated with ODN2006 (a synthetic oligonucleotide strongly activating B cells, 10 μg/mL, InvivoGen) and CD40L (1 μg/mL, R&D Systems) for 24 hours, followed by a leukocyte activation cocktail (a mixture containing Phorbol 12-Myristate 13-Acetate, ionomycin, and Brefeldin A, 2 μl/mL, BD Pharmingen) before another 4-hour culture (22). Then, the cells were permeabilized and incubated with specific antibodies. Flow cytometry was performed on a FACSAria II (BD Immunocytometry Systems) according to the manufacturer's instructions, and analyzed with FlowJo software version 7.6.1(Tree Star).

Coculture of immune cells and tumor cells

CD8+ T cells were sorted from isolated lymphocytes stained with FITC-labeled CD8 antibodies. CD20+ B and CD8+ T cells derived from the hepatocellular carcinoma margin area, together with a human hepatocellular carcinoma cell line MHCC97H (97H), were used for coculture assays. The 97H cell line was established in our institute (23, 24) from a subcutaneous xenograft of a human metastatic hepatocellular carcinoma model in nude mice, and expressed low HLA-ABC and bare HLA-DR (Supplementary Fig. S1). Briefly, 97H cells (1.0 × 104 cells/well) were seeded with CD20+ B cells (1.0 × 104 cells/well), CD8+ T cells (1.0 × 104 cells/well), or a combination of CD20+ plus CD8+ cells (5.0 × 103 cells/well for each), respectively, in 96-well plates with a mixture of Dulbecco's modified Eagle Medium (DMEM) and RPMI-1640 (1:1, Invitrogen) containing 10% FBS (Invitrogen), using 97H cells (1.0 × 104 cells/well) without any lymphocytes as control. The cocultures were incubated for 5 days, and the supernatant was collected for ELISA analysis. Tumor cell viability on each day was evaluated using the CCK-8 assay (Dojindo Inc.) according to the manufacturer's instructions. Six independent experiments with different patient samples were conducted in triplicate.

ELISA analysis

The concentration of IFN-γ in the conditioned supernatant from the coculture experiment was determined using a sandwich ELISA kit according to the manufacturer's instructions (DuoSet, R&D Systems).

Immunofluorescence

The desired tumor tissues, obtained from 12 patients with hepatocellular carcinoma and underwent hepatectomy in our institute, were embedded with OCT tissue-freezing medium (Tissue-Tek, Sakura Finetek), and stored at −80°C immediately after surgery. Tumor samples from another five patients with hepatocellular carcinoma were used for CD20+ cell sorting. MIL and peritumor-infiltrating B lymphocytes (PIL-B) were collected on slides using Cytospin 4 (Thermo Scientific). Immunofluorescence was performed as described previously (25), using anti-CD20 mouse mAb (1:200 dilution, clone L26, Dako) together with either anti-granzyme B rabbit pAb (1:50 dilution, Abcam) or anti-TRAIL (C92B9) rabbit mAb (1:500 dilution, Cell Signaling Technology). Then, two secondary antibodies, goat anti-mouse IgG-Cy3 and goat anti-rabbit IgG-FITC (Invitrogen), were applied. Images were acquired with a LSM510 Confocal Laser Scanning Microscope (Carl Zeiss).

Statistical analysis

Data were expressed as the mean ± SEM, and error bars refer to SEM in figures. The analysis of the association between variables was conducted using the Spearman ρ coefficient test, Student t test, χ2 test, Fisher exact test, or one-way ANOVA when appropriate. Univariate and multivariate analyses were based on the Cox proportional hazards regression model. Survival curves were computed using the Kaplan–Meier method (log-rank test). A two-tailed P value of 0.05 indicated a significant result. All statistical analyses were conducted with SPSS version 19.0 (IBM).

The distribution patterns of CD20+ B cells in hepatocellular carcinoma

Immunohistochemistry was performed on 120 HCC samples to elucidate the distribution of CD20+ B cells in the peritumor, tumor margin, and intratumor areas (Fig. 1A). B cells were predominately enriched in the invasive margin (median, 192.7 cells/mm2; range, 29.6–820.3 cells/mm2), whereas only small fractions of B cells sporadically infiltrated into the peritumor (PIL-Bs; median, 49.5 cells/mm2; range, 0–354 cells/mm2, P < 0.001) and intratumor (median, 12 cells/mm2; range, 0–257 cells/mm2, P < 0.001; Fig. 1B) areas. To confirm the result, flow cytometry was conducted to detect CD20+ B cells in the three microanatomic areas from 10 patients with hepatocellular carcinoma. Accordingly, the percentage of CD20+ B cells within the total CD45+ cell population in the invasive margin (9.4 ± 0.70%) was significantly higher than that in the peritumor (4.9 ± 0.57%, P = 0.001) and intratumor areas (1.8 ± 0.41%, P < 0.001), respectively (Supplementary Fig. S2). Because the intratumoral area contained the least number of B cells, whereas most B cells were enriched in the tumor margin, we consequently focused on prognostic value and functional status of MIL-Bs, with PIL-Bs as the control.

Figure 1.

Immunostaining results of infiltrating lymphocytes in hepatocellular carcinoma (HCC). A, left, a representative case of CD20 immunostaining in hepatocellular carcinoma tissue. The solid-line curve indicates the tumor–normal border, and the area between the dashed curves denotes the margin area. Magnification, ×4. Right, immunomap of the same hepatocellular carcinoma lesion. The tumor section is artificially divided into tiles (0.16 mm2 each), and colored according to CD20+ density. B, CD20+ B cells are more abundant in the margin area than in the peritumor and intratumor areas (n = 120). Lines indicate 25th, 50th, and 75th percentiles. ***, P < 0.001. C, representative hematoxylin and eosin (HE) staining of hepatocellular carcinomas with (left) or without (right) tumor capsule (top). Magnification, ×10. Also shown is the immunostaining of CD20+ B cells in cases without (middle) or with (bottom) penetration through the tumor capsule. Magnification, left, ×10; right, ×40. White lines highlight the tumor capsule. D, serial sections show hepatocellular carcinomas with simultaneously high (top) or low (bottom) densities of marginal CD20+ and CD8+ cells. Magnification, ×20. E, the density of CD20+ B cells significantly correlates with the density of CD8+ T cells in the tumor margin (n = 120; r = 0.391, P < 0.001).

Figure 1.

Immunostaining results of infiltrating lymphocytes in hepatocellular carcinoma (HCC). A, left, a representative case of CD20 immunostaining in hepatocellular carcinoma tissue. The solid-line curve indicates the tumor–normal border, and the area between the dashed curves denotes the margin area. Magnification, ×4. Right, immunomap of the same hepatocellular carcinoma lesion. The tumor section is artificially divided into tiles (0.16 mm2 each), and colored according to CD20+ density. B, CD20+ B cells are more abundant in the margin area than in the peritumor and intratumor areas (n = 120). Lines indicate 25th, 50th, and 75th percentiles. ***, P < 0.001. C, representative hematoxylin and eosin (HE) staining of hepatocellular carcinomas with (left) or without (right) tumor capsule (top). Magnification, ×10. Also shown is the immunostaining of CD20+ B cells in cases without (middle) or with (bottom) penetration through the tumor capsule. Magnification, left, ×10; right, ×40. White lines highlight the tumor capsule. D, serial sections show hepatocellular carcinomas with simultaneously high (top) or low (bottom) densities of marginal CD20+ and CD8+ cells. Magnification, ×20. E, the density of CD20+ B cells significantly correlates with the density of CD8+ T cells in the tumor margin (n = 120; r = 0.391, P < 0.001).

Close modal

In 79 of 120 cases (65.8%), a fibrous connective tissue surrounding the neoplastic mass, defined as a tumor capsule (26), was detected (Fig. 1C, top left). Interestingly, we found an uneven distribution of MIL-Bs on both sides of the tumor capsule. In some cases (54.4%, 43/79), MIL-Bs just assembled outside the tumor capsule (Fig. 1C, middle), whereas in others (45.6%, 36/79) MIL-Bs were found on both sides of the tumor capsule (Fig. 1C, bottom). It seemed that, in these 36 cases, MIL-Bs had penetrated the tumor capsule and interacted directly with tumor cells.

Serial sections were used to stain for CD20+ B and CD8+ T cells in the tumor margin. In line with a previous study (27), we found that MIL-Bs always colocalized with CD8+ T cells (Fig. 1D). A significant positive correlation (P < 0.001, r = 0.391) was revealed between the densities of the two cell types in tumor margin (Fig. 1E), which implied a potential cooperation between them in a tumor-killing effect.

The association of CD20+ cells with clinicopathologic features

We further investigated the relationship of MIL-B density with clinicopathologic features, using the median as the cutoff. Patients with high MIL-B density were prone to have small tumor size (P = 0.004) and absence of vascular invasion (P = 0.041) (Table 1). Notably, patients with MIL-Bs that had penetrated through the tumor capsule tended to have a single tumor (P = 0.014) and absence of vascular invasion (P = 0.035; Table 1). All the correlations were further recapitulated in the validation cohort (Supplementary Table S1). In contrast, PIL-B density showed no obvious associations with these clinicopathologic features except that high density of PIL-Bs was significantly correlated with history of hepatitis (P = 0.022; Table 1). However, the correlation did not reach significance in the validation cohort (P = 0.097; Supplementary Table S1).

Table 1.

Correlation between CD20+ B cells and clinicopathologic characteristics (n = 120)

MIL-BsPIL-BsPenetrationb
CharacteristicsLowHighPLowHighPNoYesP
Gender 
 Male 48 55 0.067 49 54 0.191 39 28 0.111 
 Female 12  11   
Age 
 ≤50 28 28 1.000 32 24 0.143 19 19 0.447 
 >50 32 32  28 36  24 17  
History of hepatitis 
 No 20 11 0.610 21 10 0.022 12 12 0.601 
 Yes 40 49  39 50  31 24  
Liver cirrhosis 
 No 11 14 0.500 13 12 0.822 10 0.681 
 Yes 49 46  47 48  33 29  
ALT (U/L) 
 ≤40 26 36 0.068 30 32 0.715 20 20 0.423 
 >40 34 24  30 28  23 16  
AFP (ng/mL) 
 ≤20 22 24 0.707 24 22 0.707 16 14 0.878 
 >20 38 36  36 38  27 22  
Tumor size (cm) 
 ≤5 37 51 0.004 41 47 0.215 30 28 0.422 
 >5 23  19 13  13  
Tumor number 
 Single 50 55 0.168 53 52 0.783 36 36 0.014a 
 Multiple 10    
Tumor encapsulation 
 No 22 18 0.439 17 24 0.178   NA 
 Yes 38 42  43 36     
Vascular invasion 
 No 30 41 0.041 38 33 0.353 21 26 0.035 
 Yes 30 19  22 27  22 10  
Tumor differentiation 
 I/II 42 43 0.841 44 41 0.547 29 28 0.307 
 III/IV 18 17  16 19  14  
TNM stage 
 I 32 40 0.136 38 34 0.456 23 26 0.088 
 II/III 28 20  22 26  20 10  
BCLC stage 
 0/A 35 44 0.083 44 35 0.083 26 25 0.406 
 B/C 25 16  16 25  17 11  
MIL-BsPIL-BsPenetrationb
CharacteristicsLowHighPLowHighPNoYesP
Gender 
 Male 48 55 0.067 49 54 0.191 39 28 0.111 
 Female 12  11   
Age 
 ≤50 28 28 1.000 32 24 0.143 19 19 0.447 
 >50 32 32  28 36  24 17  
History of hepatitis 
 No 20 11 0.610 21 10 0.022 12 12 0.601 
 Yes 40 49  39 50  31 24  
Liver cirrhosis 
 No 11 14 0.500 13 12 0.822 10 0.681 
 Yes 49 46  47 48  33 29  
ALT (U/L) 
 ≤40 26 36 0.068 30 32 0.715 20 20 0.423 
 >40 34 24  30 28  23 16  
AFP (ng/mL) 
 ≤20 22 24 0.707 24 22 0.707 16 14 0.878 
 >20 38 36  36 38  27 22  
Tumor size (cm) 
 ≤5 37 51 0.004 41 47 0.215 30 28 0.422 
 >5 23  19 13  13  
Tumor number 
 Single 50 55 0.168 53 52 0.783 36 36 0.014a 
 Multiple 10    
Tumor encapsulation 
 No 22 18 0.439 17 24 0.178   NA 
 Yes 38 42  43 36     
Vascular invasion 
 No 30 41 0.041 38 33 0.353 21 26 0.035 
 Yes 30 19  22 27  22 10  
Tumor differentiation 
 I/II 42 43 0.841 44 41 0.547 29 28 0.307 
 III/IV 18 17  16 19  14  
TNM stage 
 I 32 40 0.136 38 34 0.456 23 26 0.088 
 II/III 28 20  22 26  20 10  
BCLC stage 
 0/A 35 44 0.083 44 35 0.083 26 25 0.406 
 B/C 25 16  16 25  17 11  

Abbreviations: ALT, alanine aminotransferase; AFP, alpha-fetoprotein; TNM, tumor–node–metastasis; BCLC, Barcelona Clinic Liver Cancer; NA, not applicable.

aFisher exact tests; χ2 test for all the other analyses.

bχ2 test for penetration was only applied in patients with tumor capsule (n = 79).

Prognostic significance of infiltrating CD20+ B cells

The 1-, 3-, and 5-year OS and RFS rates of the 120 patients with hepatocellular carcinoma were 89%, 66%, and 44% and 77%, 54%, and 42%, respectively. In univariate analyses, conventional clinicopathologic features that correlated with a dismal OS and RFS were high alanine aminotransferase (ALT) level, large tumor size, tumor multiplicity, absence of encapsulation, presence of vascular invasion, and advanced tumor stages (Supplementary Table S2). As expected, a significant positive correlation between the density of MIL-Bs and OS (P < 0.001) or RFS (P < 0.001) was detected (Fig. 2A). Patients with above-median level of MIL-Bs had a significantly higher 5-year OS (58% vs. 30% for high or low MIL-Bs, respectively) and RFS rates (63% vs. 22% for high or low MIL-Bs, respectively). However, the density of PIL-Bs was associated with neither OS (P = 0.689) nor RFS (P = 0.572; Fig. 2B). Intriguingly, the penetration of MIL-Bs through the tumor capsule indicated a markedly better OS (5-year rate, 69% vs. 36% for penetration and no penetration, respectively; P = 0.003) and RFS (5-year rate, 64% vs. 28% for penetration and no penetration, respectively; P = 0.003; Fig. 2C).

Figure 2.

Kaplan–Meier curves of OS and RFS according to the densities of: A, MIL-Bs; B, PIL-Bs; C, penetration of MIL-Bs through tumor capsule; and D, the combination of marginal CD20+ and CD8+ cell densities in the margin area. High density of MIL-Bs, the presence of penetration of MIL-Bs and simultaneously high densities of CD20+ and CD8+ cells are significantly associated with both prolonged overall and recurrence-free survival. P values are based on log-rank test.

Figure 2.

Kaplan–Meier curves of OS and RFS according to the densities of: A, MIL-Bs; B, PIL-Bs; C, penetration of MIL-Bs through tumor capsule; and D, the combination of marginal CD20+ and CD8+ cell densities in the margin area. High density of MIL-Bs, the presence of penetration of MIL-Bs and simultaneously high densities of CD20+ and CD8+ cells are significantly associated with both prolonged overall and recurrence-free survival. P values are based on log-rank test.

Close modal

Then, variables with P < 0.1 in univariate analysis (included tumor encapsulation with a borderline significance for RFS; P = 0.115) were adopted as covariates in multivariate Cox proportional hazards analyses. Multivariate analyses revealed that the association of high MIL-B density with better OS [HR = 0.44; 95% confidence interval (CI) = 0.26–0.76; P = 0.003] or RFS (HR = 0.49; 95% CI = 0.28–0.86; P = 0.012; Table 2) was independent of ALT level, tumor encapsulation, tumor size, tumor number, and vascular invasion. In addition, the penetration of MIL-Bs through the tumor capsule independently indicated prolonged OS (HR = 0.44; 95% CI = 0.20–0.97; P = 0.042) as well as RFS (HR = 0.47; 95% CI = 0.23–0.97; P = 0.040; Table 2).

Table 2.

Multivariate analyses of factors associated with OS and RFS

OSRFS
VariablesHR (95% CI)PHR (95% CI)P
All patients in the initial cohort (n = 120) 
 ALT (U/L; ≤40 vs. >40)  NS  NS 
 Size (cm; ≤5 vs. >5)  NS 1.73 (1.01–2.97) 0.044 
 Tumor number (single vs. multiple)  NS  NS 
 Tumor encapsulation (no vs. yes) 0.51 (0.31–0.85) 0.009  NS 
 Vascular invasion (no vs. yes) 1.88 (1.13–3.15) 0.016 2.45 (1.47–4.08) 0.001 
 TNM stage (I vs. II/III)  NA  NA 
 BCLC stage (0/A vs. B/C)  NA  NA 
 MIL-Bs (low vs. high) 0.44 (0.26–0.76) 0.003 0.49 (0.28–0.86) 0.012 
Patients with tumor capsule in the initial cohort (n = 79) 
 ALT (U/L; ≤40 vs. >40) 2.89 (1.36–6.16) 0.006  NS 
 Size (cm; ≤5 vs. >5)  NS  NS 
 Tumor number (single vs. multiple) 3.70 (1.38–9.93) 0.010 3.11 (1.10–8.79) 0.032 
 Tumor encapsulation (no vs. yes)  NA  NA 
 Vascular invasion (no vs. yes)  NS 2.21 (1.07–4.58) 0.032 
 TNM stage (I vs. II/III)  NA  NA 
 BCLC stage (0/A vs. B/C)  NA  NA 
 Penetration (no vs. yes) 0.44 (0.20–0.97) 0.042 0.47 (0.23–0.97) 0.040 
All patients in the validation cohort (n = 200) 
 ALT (U/L; ≤40 vs. >40)  NS  NS 
 Size (cm; ≤5 vs. >5) 1.68 (1.16–2.42) 0.006  NS 
 Tumor number (single vs. multiple) 2.03 (1.25–3.30) 0.004 1.81 (1.03–3.18) 0.038 
 Tumor encapsulation (no vs. yes)  NS  NS 
 Vascular invasion (no vs. yes) 1.83 (1.27–2.64) 0.001 1.94 (1.29–2.93) 0.002 
 TNM stage (I vs. II/III)  NA  NA 
 BCLC stage (0/A vs. B/C)  NA  NA 
 MIL-Bs (low vs. high) 0.53 (0.37–0.77) 0.001 0.60 (0.40–0.91) 0.015 
Patients with tumor capsule in the validation cohort (n = 93) 
 ALT (U/L; ≤40 vs. >40)  NS  NS 
 Size (cm; ≤5 vs. >5) 1.95 (1.14–3.32) 0.014 1.87 (1.01–3.44) 0.046 
 Tumor number (single vs. multiple)  NS  NS 
 Tumor encapsulation (no vs. yes)  NA  NA 
 Vascular invasion (no vs. yes)  NS  NS 
 TNM stage (I vs. II/III)  NA  NA 
 BCLC stage (0/A vs. B/C)  NA  NA 
 Penetration (no vs. yes) 0.57 (0.34–0.97) 0.038 0.44 (0.23–0.83) 0.011 
OSRFS
VariablesHR (95% CI)PHR (95% CI)P
All patients in the initial cohort (n = 120) 
 ALT (U/L; ≤40 vs. >40)  NS  NS 
 Size (cm; ≤5 vs. >5)  NS 1.73 (1.01–2.97) 0.044 
 Tumor number (single vs. multiple)  NS  NS 
 Tumor encapsulation (no vs. yes) 0.51 (0.31–0.85) 0.009  NS 
 Vascular invasion (no vs. yes) 1.88 (1.13–3.15) 0.016 2.45 (1.47–4.08) 0.001 
 TNM stage (I vs. II/III)  NA  NA 
 BCLC stage (0/A vs. B/C)  NA  NA 
 MIL-Bs (low vs. high) 0.44 (0.26–0.76) 0.003 0.49 (0.28–0.86) 0.012 
Patients with tumor capsule in the initial cohort (n = 79) 
 ALT (U/L; ≤40 vs. >40) 2.89 (1.36–6.16) 0.006  NS 
 Size (cm; ≤5 vs. >5)  NS  NS 
 Tumor number (single vs. multiple) 3.70 (1.38–9.93) 0.010 3.11 (1.10–8.79) 0.032 
 Tumor encapsulation (no vs. yes)  NA  NA 
 Vascular invasion (no vs. yes)  NS 2.21 (1.07–4.58) 0.032 
 TNM stage (I vs. II/III)  NA  NA 
 BCLC stage (0/A vs. B/C)  NA  NA 
 Penetration (no vs. yes) 0.44 (0.20–0.97) 0.042 0.47 (0.23–0.97) 0.040 
All patients in the validation cohort (n = 200) 
 ALT (U/L; ≤40 vs. >40)  NS  NS 
 Size (cm; ≤5 vs. >5) 1.68 (1.16–2.42) 0.006  NS 
 Tumor number (single vs. multiple) 2.03 (1.25–3.30) 0.004 1.81 (1.03–3.18) 0.038 
 Tumor encapsulation (no vs. yes)  NS  NS 
 Vascular invasion (no vs. yes) 1.83 (1.27–2.64) 0.001 1.94 (1.29–2.93) 0.002 
 TNM stage (I vs. II/III)  NA  NA 
 BCLC stage (0/A vs. B/C)  NA  NA 
 MIL-Bs (low vs. high) 0.53 (0.37–0.77) 0.001 0.60 (0.40–0.91) 0.015 
Patients with tumor capsule in the validation cohort (n = 93) 
 ALT (U/L; ≤40 vs. >40)  NS  NS 
 Size (cm; ≤5 vs. >5) 1.95 (1.14–3.32) 0.014 1.87 (1.01–3.44) 0.046 
 Tumor number (single vs. multiple)  NS  NS 
 Tumor encapsulation (no vs. yes)  NA  NA 
 Vascular invasion (no vs. yes)  NS  NS 
 TNM stage (I vs. II/III)  NA  NA 
 BCLC stage (0/A vs. B/C)  NA  NA 
 Penetration (no vs. yes) 0.57 (0.34–0.97) 0.038 0.44 (0.23–0.83) 0.011 

Abbreviations: ALT, alanine aminotransferase; BCLC, Barcelona Clinic Liver Cancer; CI, confidence interval; MIL-Bs, margin-infiltrating B cells; NA, not applicable; NS, not significant; OS, overall survival; RFS, recurrence-free survival.; TNM, tumor–node–metastasis.

Considering the positive correlation between CD20 and CD8 in the tumor margin, we further evaluated their combined influence on patient outcome. Patients were classified into four groups, using their median as the cutoff: I, CD20 low and CD8 low (n = 36); II, CD20 low and CD8 high (n = 24); III, CD20 high and CD8 low (n = 24); and IV, CD20 high and CD8 high (n = 36). Differences in both OS (P = 0.002) and RFS (P = 0.001) were significant among the four groups (Fig. 2D). The 5-year OS (RFS, in brackets) rates were 29% (21%), 32% (24%), 47% (50%), and 64% (69%) for groups I, II, III, and IV, respectively. Thus, the data further supported the hypothesis that the cooperation between CD20+ and CD8+ cells could potently enhance the antitumor effect in hepatocellular carcinoma.

Independent validation

We further validated the prognostic value of MIL-Bs in another cohort of 200 hepatocellular carcinoma cases. In the validation cohort, a high density of MIL-Bs indicated significantly prolonged OS (5-year rate 41% vs. 19%, P < 0.001) and RFS (5-year rate 55% vs. 33%, P = 0.007), compared with patients with low MIL-B density (Supplementary Fig. S3A). Also, the penetration of MIL-Bs through the tumor capsule predicted better OS (5-year rate, 62% vs. 31% for penetration and no penetration, respectively, P = 0.004) and RFS (5-year rate 56% vs. 27% for penetration and no penetration, respectively, P = 0.005; Supplementary Fig. S3B). In particular, the multivariate Cox regression model adjusted to established clinical factors authenticated the density and penetration of MIL-Bs as independent prognosticators (P < 0.05) for patients with hepatocellular carcinoma (Table 2).

The phenotypes of MIL-Bs

Flow cytometry showed that PIL-Bs had a naïve phenotype (IgD+IgM+IgG), whereas MIL-Bs displayed an active mature phenotype (IgDIgM−/lowIgG+; Fig. 3A). This antigen-dependent switch from IgM and IgD to IgG production is a well-known feature of B-cell maturation (28). In addition, MIL-Bs were CD27 and CD38, indicative of an atypical memory phenotype (described in the discussion; Fig. 3A; ref. 27).

Figure 3.

Flow cytometric analyses of infiltrating B cells. A, representative expressions of surface markers on cells that were gated on CD20+ cells. Cells were stained with indicated antibodies or isotype-matched controls (gray filled area). Red line, MIL-Bs; Blue line, PIL-Bs (n = 10). B, Be-1/Be-2 cytokine expression profile in stimulated MIL-Bs using the same gating strategy in (A; n = 10). For cytokine detection, CD20+ B cells were stimulated with ODN2006 (10 μg/mL, InvivoGen) and CD40L (1 μg/mL, R&D Systems) for 24 hours, followed by the addition of a leukocyte activation cocktail (2 μl/mL, BD Pharmingen) before another 4-hour culture. Quadrant gates were established on the basis of isotype-matched controls, and the number in the quadrant indicates the percentage of cells.

Figure 3.

Flow cytometric analyses of infiltrating B cells. A, representative expressions of surface markers on cells that were gated on CD20+ cells. Cells were stained with indicated antibodies or isotype-matched controls (gray filled area). Red line, MIL-Bs; Blue line, PIL-Bs (n = 10). B, Be-1/Be-2 cytokine expression profile in stimulated MIL-Bs using the same gating strategy in (A; n = 10). For cytokine detection, CD20+ B cells were stimulated with ODN2006 (10 μg/mL, InvivoGen) and CD40L (1 μg/mL, R&D Systems) for 24 hours, followed by the addition of a leukocyte activation cocktail (2 μl/mL, BD Pharmingen) before another 4-hour culture. Quadrant gates were established on the basis of isotype-matched controls, and the number in the quadrant indicates the percentage of cells.

Close modal

B cells can influence antitumor immunity by serving as APCs, which may involve MHC and costimulatory molecules. We found that MIL-Bs and PIL-Bs expressed similar intensities of MHC class I (HLA-A, B and C), class II (HLA-DR) molecules, and CD40. However, MIL-Bs expressed significantly higher levels of costimulatory molecules B7-1 (CD80; P < 0.001) and B7-2 (CD86; P < 0.001) compared with PIL-Bs (Fig. 3A). The data suggested that, compared with PIL-Bs, the MIL-Bs were potentially more potent in presenting antigen and activating T cells in the tumor microenvironment (29).

The antitumor effect of MIL-Bs

B cells can be directed to secrete polarized groups of cytokines. Regulatory B cells are characterized by secreting IL-10 or TGF-β1, whereas effector B-cell populations are grouped into two categories: (i) Be-1 cells producing cytokines such as IFN-γ, IL-12, and TNF-α, and (ii) Be-2 cells releasing IL-2, IL-4, TNF-α, and IL-6 (30). Flow cytometric analysis showed that MIL-Bs mainly produced IFN-γ (8.52 ± 0.44%) and IL-12p40 (2.07 ± 0.11%) rather than IL-2, IL-4, IL-6, or IL-10 (0.05%–0.5%; Fig. 3B), indicating a Be-1 phenotype. However, PIL-Bs scarcely produced these cytokines (data not shown). All the data supported the notion that MIL-Bs could facilitate the Th1 immune response, leading to strong antitumor activity.

In vitro assays showed that the viability of 97H cells was significantly inhibited, when cocultured with either CD8+ T cells or MIL-Bs, suggesting that B cells might exert direct killing effects similar to CD8+ T cells. Moreover, the cooperation of MIL-Bs and CD8+ T cells was substantiated by the findings that the combination of the two cell types caused a steeper decrease in tumor cell viability during the first 3 days (P < 0.05; Fig. 4A). In addition, the inhibiting effect of the two cell types used in combination lasted for as long as 5 days compared with merely 3 days for each type used alone (data not shown). Similarly, ELISAs revealed that the IFN-γ level in the supernatant of coculture group with both MIL-Bs and CD8+ T cells (65.26 ± 5.54 pg/mL) was significantly higher than the groups of either type of lymphocyte used alone (P < 0.05; Fig. 4B), supporting a synergistic effect of the two cell types.

Figure 4.

Functional assays for tumor-killing effect of MIL-Bs. A, results of 97H cell viability from cocultures performed in groups as indicated. The percentage of viability was plotted relative to control 97H cells. Difference between groups was determined by one-way ANOVA. **, P < 0.01; ***, P < 0.001. Six independent experiments with different patient samples were conducted in triplicate. B, IFN-γ production was measured by ELISA in the supernatant of the coculture groups. Results were similar on day 1, 2, or 3. *, P < 0.05; **, P < 0.01. Six independent experiments were conducted in triplicate. C and D, immunofluorescence images of margin tissue (n = 12) and sorted CD20+ B cells (n = 5), showed the coexpression of CD20 (red) with granzyme B (green) or TRAIL (green). The top panels in (C) and (D) are for tissue immunofluorescence (Magnification, ×10; Yellow line, the tumor–normal border), whereas the middle and bottom are for sorted MIL-Bs and PIL-Bs, respectively (Magnification, × 100). The low power micrographs show that the percentages of MIL-Bs positive for granzyme B and TRAIL are 43.2 ± 4.3% and 36.2 ± 3.8%, respectively (n = 5), and both percentages of PIL-Bs positive for granzyme B and TRAIL are below 10% (n = 5).

Figure 4.

Functional assays for tumor-killing effect of MIL-Bs. A, results of 97H cell viability from cocultures performed in groups as indicated. The percentage of viability was plotted relative to control 97H cells. Difference between groups was determined by one-way ANOVA. **, P < 0.01; ***, P < 0.001. Six independent experiments with different patient samples were conducted in triplicate. B, IFN-γ production was measured by ELISA in the supernatant of the coculture groups. Results were similar on day 1, 2, or 3. *, P < 0.05; **, P < 0.01. Six independent experiments were conducted in triplicate. C and D, immunofluorescence images of margin tissue (n = 12) and sorted CD20+ B cells (n = 5), showed the coexpression of CD20 (red) with granzyme B (green) or TRAIL (green). The top panels in (C) and (D) are for tissue immunofluorescence (Magnification, ×10; Yellow line, the tumor–normal border), whereas the middle and bottom are for sorted MIL-Bs and PIL-Bs, respectively (Magnification, × 100). The low power micrographs show that the percentages of MIL-Bs positive for granzyme B and TRAIL are 43.2 ± 4.3% and 36.2 ± 3.8%, respectively (n = 5), and both percentages of PIL-Bs positive for granzyme B and TRAIL are below 10% (n = 5).

Close modal

Meanwhile, immunofluorescence on tissues and sorted cells demonstrated obviously enhanced expression of granzyme B and TRAIL in MIL-Bs (Fig. 4C and D), both of which could have direct cytotoxicity against tumor cells through antibody-independent mechanisms. In contrast, PIL-Bs expressed low levels of granzyme B and TRAIL (Fig. 4C and D). We further confirmed the results by flow cytometry. The percentages of granzyme B+ (42.2 ± 3.1%) and TRAIL+ cells (25.7 ± 1.7%) in total CD20+ cells were significantly higher in MIL-Bs than in PIL-Bs (about 4-fold in granzyme B+ cells, P < 0.001 and 3-fold in TRAIL+ cells, P = 0.001, respectively, n = 5; Supplementary Fig. S4). The data implied that the microenvironment around the tumor margin could facilitate the activation and tumor-killing potential of CD20+ cells.

Most studies on the phenotype and functional activity of infiltrating lymphocytes in tumors have been focused on T lineage, while considerably less is known about infiltrating B cells. Herein, for the first time, we presented a detailed investigation of the distribution, phenotype, and function of infiltrating CD20+ B cells in human hepatocellular carcinoma. We observed that the CD20+ B cells were concentrated around the tumor deposit and formed a dense cell layer in the invasive margin area, which simultaneously contained a high level of CD8+ T cells. Importantly, we identified and validated the presence of MIL-Bs and their penetration through the tumor capsule as independent favorable prognostic factors for patient survival and tumor recurrence.

These findings corresponded with several previous reports that infiltrating B cells were prevalent in human cancers, recognizing a wide variety of tumor antigens, associating closely with T cells and other immune cells, and correlating with favorable outcomes (31, 32). However, there were conflicting data suggesting that B cells had a negative effect on protective antitumor responses and might even facilitate tumor progression (33, 34). For instance, Olkhanud and colleagues (34) identified a new subset of B cells, tumor-evoked regulatory B cells, which resembled Be-2 phenotype and promoted breast cancer metastasis by converting resting CD4+ T cells to T-regulatory cells. One reason for this discrepancy may lie in the functional statuses of B cells in different contexts, as Be-1 and Be-2 have the capacity to regulate Th1 or Th2 differentiation by secreting polarized arrays of cytokines (35, 36).

To understand how in situ MIL-Bs accumulation influenced tumor immunity, we characterized the phenotype of these B cells. First, MIL-Bs displayed an active mature phenotype (IgDIgM−/lowIgG+), whereas PIL-Bs exhibited a naïve phenotype (IgD+IgM+IgG). This phenomenon was regarded as that the MIL-Bs were undergoing Ig class switching in response to antigen exposure and were gradually activated in the tumor margin. Second, MIL-Bs expressed surface markers that were characteristic of APCs, including MHC I, MHC II, CD40, CD80, and CD86, so that MIL-Bs were capable of recognizing and presenting tumor-associated antigens. The antigen spectrum of the B-cell antigen receptor (BCR) is not limited to peptide antigens but also includes carbohydrates, phospholipids, nucleic acids, and larger structures, such as virus particles. Because BCR-mediated endocytosis could concentrate small quantities of specific antigens and amplify the responses to tumor antigens expressed at low levels, B cells could be more easily manipulated to generate an ideal antitumor response. Third, the majority of the MIL-Bs failed to express CD38, further indicative of a memory phenotype (37). In addition, the lack of CD27 expression of MIL-Bs suggested that they might belong to an atypical memory B cell (38). CD38 is a biomarker of plasma cells, and CD27 is a conventional memory B-cell marker with the capacity to bind certain surface molecules on activated T helper cells that induce Ig secretion in humoral immunity (39). It has been documented that the deficiency of CD38 and CD27 expressions on B cells might restrict antibody production but promote cytolytic antitumor responses (40, 41). Coincidentally, the CD20+ tumor-infiltrating lymphocyte with similar atypical memory phenotype was observed in ovarian cancer as well (27). Furthermore, we demonstrated that MIL-Bs produced high levels of IFN-γ and IL-12p40 instead of IL-2, IL-4, IL-6, and IL-10. Taken together, we proposed that the MIL-Bs in hepatocellular carcinoma might belong to the Be-1 subset, which mainly participates in cellular-mediated antitumor immunity.

We also observed the intense expression of granzyme B and TRAIL on MIL-Bs in hepatocellular carcinoma, which suggested MIL-Bs might be involved in direct cytotoxicity. The secretion of granzyme B by human B cells required stimulation with IL-21 (42), whereas IFN-γ stimulation or a Toll-like receptor agonist was necessary for potent TRAIL signaling (43). Therefore, the cytokine milieu in the hepatocellular carcinoma invasive front might be conducive to the production of granzyme B and TRAIL, and program MIL-Bs toward a status capable of killing tumor cells directly.

Survival analyses showed that the patient containing high levels of both MIL-Bs and CD8+ T cells had a superior prognosis to those with either cell type alone, suggesting cooperative interactions between CD20+ and CD8+ cells. The close proximity and the similar cooperative effect between tumor-infiltrating CD20+ and CD8+ lymphocytes were seen in ovarian cancer (27) as well. In coculture experiments, a combination of the two cell types induced a stronger and longer lasting inhibition of tumor growth than either of the two types used alone. Similarly, the combined use of the two cell types increased IFN-γ production to the largest extent. As such, a strong cooperation between the two types of lymphocytes made it valuable to explore the mechanisms underlying the interaction occurring in the invasive margin area.

In conclusion, we demonstrated that an increased density of MIL-Bs was associated with better survival and reduced recurrence in patients who had hepatocellular carcinoma. The potential mechanisms included MIL-Bs serving as APC, stimulating CD8+ T cells by releasing proinflammatory cytokines, and exerting a direct killing effect. Thus, an improved understanding of the mechanism of MIL-B recruitment to the tumor, MIL-B target antigens, and interactions of MIL-Bs with other immune cells might facilitate the design of more effective immunotherapies for hepatocellular carcinoma.

No potential conflicts of interest were disclosed.

Conception and design: J.-Y. Shi, J. Fan

Development of methodology: J.-Y. Shi, Z.-H. Min, Z.-B. Ding

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Q. Gao, Z.C. Wang, J. Zhou, X. Wang, Y.-H. Shi, G.-M. Shi, A.-W. Ke, K. Song

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.-Y. Shi, Q. Gao, Z.C. Wang, Z.-H. Min, Z. Dai, J. Fan

Writing, review, and/or revision of the manuscript: J.-Y. Shi, Q. Gao, Z.C. Wang

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.-Y. Shi, J. Zhou, Z.-B. Ding, S.-J. Qiu, J. Fan

Study supervision: S.-J. Qiu, J. Fan

This work was financially supported by the Major Program of the NSFC (grant no. 81030038), National Key Sci-Tech Project (grant no. 2012ZX10002011-002), China National Funds for Distinguished Young Scientists (grant no. 812250125), National Natural Science Foundation of China (grant nos. 81071992 and 81272730), and the Fok Ying–Tong Education Foundation (grant no. 132029).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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