Clinical significance of tumor-infiltrating immune cells focusing on BTLA and Cbl-b in patients with gallbladder cancer.

The host immune system plays a significant role in tumor control, although most cancers escape immune surveillance through a variety of mechanisms. The aim of the present study was to evaluate the clinicopathological significance of a novel co-inhibitory receptor, B and T lymphocyte attenuator (BTLA), the anergy cell marker Casitas-B-lineage lymphoma protein-b (Cbl-b), and clinical implications of tumor-infiltrating immune cells in gallbladder cancer (GBC) tissues. We investigated 211 cases of GBC, 21 cases of chronic cholecystitis (CC), and 11 cases of xanthogranulomatous cholecystitis (XGC) using immunohistochemistry to detect tissue-infiltrating immune cells and their expression of BTLA and Cbl-b, and carried out correlation and survival analyses. The density of infiltrating T cells was significantly higher in CC and XGC than in GBC. The density ratio of BTLA(+) cells to CD8(+) T cells (BTLA/CD8) and that of Cbl-b(+) cells to CD8(+) T cells (Cbl-b/CD8) were significantly higher in GBC than in CC and XGC. The FOXP3/CD4, BTLA/CD8, and Cbl-b/CD8 ratios were significantly correlated with each other, and also with malignant phenotypes. Survival analyses revealed that a lower density of tumor-infiltrating CD8(+) cells, and higher Foxp3/CD4, BTLA/CD8, and Cbl-b/CD8 ratios were significantly associated with shorter overall survival and disease-free survival in GBC patients. Multivariate analyses showed that M factor, perineural invasion, BTLA/CD8, and Cbl-b/CD8 were closely associated with shorter overall survival. These findings suggest that higher ratios of BTLA/CD8 and Cbl-b/CD8 are independent indicators of unfavorable outcome in GBC patients, and that upregulation of BTLA in cancer tissues is involved in inhibition of antitumor immunity.

Gallbladder cancer (GBC) is the most common malignant biliary neoplasm and the seventh most common gastrointestinal cancer.1 Complete surgical resection is the standard treatment for patients with localized disease, and the only potentially definitive curative therapy.2, 3 However, because most cases of GBC are diagnosed at an advanced stage, when the disease is not amenable to surgical resection, this malignancy is highly lethal, with a 5‐year survival rate of <5% for such patients.4, 5 Therefore, a more thorough understanding of GBC is essential for the development of new therapeutic strategies.

Based on a growing body of evidence from both animal and human models, it is generally accepted that naturally occurring immunity can play a significant role in the control of tumor development and progression.6 Most clinically evident cancers exhibit immune escape, where the cancer microenvironment can induce immune tolerance through a variety of mechanisms, such as the production of soluble immunosuppressive factors and the recruitment of suppressor immune cells.7, 8 Elucidation of the molecular and cellular events responsible for tumor immune escape is essential in order to make anticancer therapy truly effective in a clinical setting.9 Tumor‐infiltrating immune cells are one of the representative cellular components of host antitumor immune responses and tumor immune escape. Tumor‐infiltrating immune cells are composed of different cell subsets, which determine the overall protumor or antitumor characteristics. For example, a high proportion of CD8+ T cells infiltrating the cancer tissue can be a favorable prognostic indicator in colorectal,10, 11 ovarian,12 esophageal,13 liver,14 and pancreatic15, 16 cancers. In contrast, patients whose cancers show marked infiltration of regulatory T cells tend to have a poorer prognosis in several types of cancer.17, 18, 19 Two groups have studied the tumor‐infiltrating immune cells in GBC using small numbers of cases (45 cases20 and 69 cases21), and showed that tumor‐infiltrating CD8+ T cells and CD4+ T cells were indicators of favorable prognosis, whereas tumor‐infiltrating FOXP3+ cells or CD20+ B cells had no significant impact on outcome.

In addition to immunosuppressive cells, tumor cells deploy distinct mechanisms to evade immune attack, including expression of ligands for co‐inhibitory receptors on T cells.22 Recently, the distinct role of co‐inhibitory receptors, such as CTL‐associated protein 4 (CTLA‐4) and programmed cell death 1 (PD‐1), in this process has been extensively investigated.23 The function of antigen‐specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of these inhibitory receptors. Expression of these receptors has been linked to functional impairment of T cells in cancer, and therapeutic blockade of these two pathways has shown clinical promise. Antagonist antibodies have been developed in order to overcome immune evasion, and anti‐CTLA‐4 and anti‐PD1 antibodies have been tested in clinical trials with encouraging results.24, 25, 26, 27 Anti‐CTLA4 antibody was the first agent shown to confer a survival benefit on patients with advanced melanoma, and was approved by the US FDA in 2010.

B and T lymphocyte attenuator (BTLA) has been identified as a novel co‐inhibitory receptor, expressed by a majority of lymphocytes, and shows structural and functional similarities to CTLA‐4 and PD‐1.28 Because tumor‐infiltrating immune cells express multiple co‐inhibitory receptors, it is assumed that dual or triple blockade of co‐inhibitory receptors will enhance antitumor immunity. Indeed, combined blockade of the PD‐1/programmed death‐ligand 1 (PD‐L1) and CTLA‐4 pathways, and that of the PD‐1/PD‐L1 and lymphocyte activation gene 3 (LAG3) pathways, has been shown to enhance antitumor effects in a human clinical study and animal model studies.29, 30, 31 Thus, BTLA is expected to be a new target for interventions aimed at reversal of immune evasion and boosting of antitumor immunity in cancer patients. Expression of BTLA has been reported in B‐cell small lymphocytic lymphoma/chronic lymphocytic leukemia cells and gastric cancer cells.32, 33 However, there are currently no data on the immunohistochemical expression of BTLA in the microenvironment of human tumors, and the clinical significance of BTLA expression in tumor‐infiltrating immune cells remains to be identified. It have also been reported that BTLA plays a critical role in the induction of peripheral T cell anergy in vivo.34 T cell anergy is also widely accepted as an important mechanism of tumor immune escape,35 although no previous studies have shown anergy cells in tissues. Casitas–B‐lineage lymphoma protein‐b (Cbl‐b), an E3 ubiquitin ligase, is critical for establishing the threshold for T cell activation and for induction of T cell anergy.36, 37

The aim of this study was to evaluate the clinicopathological impact of BTLA as well as the clinical implications of tumor‐infiltrating immune cells in patients with GBC to assess their potential as new targets for cancer immunotherapy.

Materials and Methods

Study population

Clinical and pathologic data and the specimens used for immunohistochemical analysis were obtained through a detailed retrospective review of the medical records of all 211 patients with GBC who had undergone surgical resection between 1985 and 2012 at the National Cancer Center Hospital (Tokyo, Japan). All of the patients included in this study underwent macroscopic curative resection, and all had primary carcinomas of the gallbladder. Neuroendocrine neoplasms were excluded. The median follow‐up period was 28 months (range, 1–289 months) for the patients overall: 89 patients (42.2%) were alive, 84 (39.8%) had died because of GBC, and 38 (18.0%) had died of other causes. During the study period, adjuvant chemotherapy was not carried out. In addition, we analyzed cases of chronic cholecystitis (CC) (n = 21) and xanthogranulomatous cholecystitis (XGC) (n = 11) as controls for evaluating the significance of tumor‐infiltrating immune cells.

This study was approved by the Institutional Review Board of the National Cancer Center, Japan. Informed consent was obtained from all participants involved in the study, and all clinical investigations were carried out in line with the principles of the Declaration of Helsinki.

Pathological examination

All of the carcinomas were examined pathologically and classified according to the World Health Organization classification,38 Union for International Cancer Control TNM classification,39 and the Japanese Society of Biliary Surgery classification of biliary tract carcinoma.40 Tumors were staged and the histopathologic variables (histopathological grading, lymphatic, venous, and perineural invasion) were evaluated and described in accordance with their classifications.39, 40

Immunohistochemistry

Immunohistochemistry was carried out on formalin‐fixed, paraffin‐embedded tissue sections using the avidin–biotin complex method as described previously.41 We used 4‐μm‐thick serial sections of representative blocks with antibodies against the following: CD3 (PS1; 1:100) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), CD4 (368; 1:100) and CD8 (4B11; 1:200) from Leica Microsystems (Newcastle‐upon‐Tyne, UK), FOXP3 (42; 1:100) produced in house,18 BTLA (HPA047211; 1:500) from Atlas Antibodies (Stockholm, Sweden), and Cbl‐b (246C5A; 1:50) from Abcam (Cambridge, UK). Immunohistochemistry without the primary antibody was used as a negative control.

Double immunostaining

We carried out double staining on formalin‐fixed paraffin‐embedded sections. First, the 4‐μm‐thick sections were immunostained using anti‐BTLA antibody or anti‐Cbl‐b antibody as the primary antibody, and visualized with 3,3′‐diaminobenzidine. After the tissue sections had been treated with glycine–HCl (pH 2.5), they were subjected to immunofluorescence staining using antibodies against each of the following antigens: CD1a (O10, Lab Vision, Fremont, CA, USA), CD3, CD4, CD8, CD14 (7, Leica Microsystems), CD20 (L26, DAKO), CD56 (1B6, Leica Microsystems), CD68 (KP1, DAKO), CD207 (12D6, Leica Microsystems), CD208 (104.G4, Immunotech, Fullerton, CA, USA), FOXP3, BTLA, and Cbl‐b. Immunostained tissue sections were analyzed with a confocal microscope (LSM5 Pascal; Carl Zeiss, Jena, Germany) equipped with a 15‐mW Kr/Ar laser.

Quantitative evaluation of tumor‐infiltrating T cell subsets, BTLA‐positive cells, and Cbl‐b‐positive cells

After immunohistochemistry, the microscopic images were imported as digital photo files using a NanoZoomer Digital Pathology system (Hamamatsu Photonics, Hamamatsu, Japan), and the density of the immunolabeled cells was analyzed using the image analysis software, Tissue Studio (Definiens, Munich, Germany). We manually selected one area as region of interest (ROI), in which the CD3‐labeled T cells had infiltrated into the tumor most densely in the specimen, when we checked it in low‐power view. In each individual case, the same ROI was applied to all the other immunostained images. The immunolabeled cells inside the ROI were automatically counted on the basis of staining intensity. In each analysis we confirmed that the immunohistochemically positive lymphocytes were appropriately detected. The density of positive cells was calculated by dividing their number by the ROI area (cells/μm2). Also, we calculated the density ratio of FOXP3 to CD4 (FOXP3/CD4), that of BTLA to CD3 or CD8 (BTLA/CD3, BTLA/CD8), and that of Cbl‐b to CD3 or CD8 (Cbl‐b/CD3, Cbl‐b/CD8). For survival and correlation analyses, patients were divided into two groups showing high and low cell infiltration, using the median value as a cut‐off.

Statistical analysis

We expressed continuous data as median and range and compared them using the Mann–Whitney U‐test. We compared categorical data by Pearson's X2 or Fisher's exact test, as appropriate. We constructed survival curves by the Kaplan–Meier method and compared them using the log–rank test. We calculated the length of overall survival (OS) from the date of surgical resection to the date of death from any cause, calculated the length of disease‐free survival (DFS) from the date of surgical resection to the date of the first radiologic findings for recurrence, and censored those on the date of the last follow‐up. To evaluate the prognostic significance of tumor‐infiltrating lymphocytes and cells expressing inhibitory molecules in patients with GBC, univariate and multivariate Cox analyses were applied. The factors found to be significant by univariate analysis were subjected to multivariate analysis. P < 0.05 was considered to denote statistical significance for all analyses. Statistical analyses were carried out using spss version 20.0 (SPSS, Chicago, IL, USA).

Results

Immunophenotype of BTLA+ cells and Cbl‐b+ cells

To examine the immunophenotype of BTLA+ cells and Cbl‐b+ cells, double immunostaining was carried out (Fig. 1). BTLA was present in a proportion of CD3+ T cells, CD4+ T cells, CD8+ T cells, CD20+ B cells, CD14+ monocytes, CD68+ macrophages, CD1a+ dendritic cells (DCs), CD207+ DCs, or CD208+ DCs, although the majority of BTLA+ cells were CD4+. In contrast, no expression of BTLA was found in FOXP3+ cells, CD56+ natural killer (NK) cells, or Cbl‐b+ cells. Cbl‐b was expressed in a small proportion of CD3+ T cells, CD4+ T cells, CD8+ T cells, Foxp3+ cells, and CD20+ B cells, and was not expressed in CD56+ NK cells, CD14+ monocytes, CD68+ macrophages, or DCs. There were no BTLA+Cbl‐b+ cells in the cancer tissues, although T cells that had infiltrated into cancer cell nests and become attached to cancer cells were often positive for Cbl‐b, and sometimes for BTLA. No tumor cells expressed BTLA or Cbl‐b in our series.

Figure 1

Double immunostaining features in gallbladder cancer samples. (a) Most B and T lymphocyte attenuator (BTLA)+ cells (black) are CD4+ T cells (red). (b, c) BTLA (black) staining is sometimes present in CD8+ T cells (red) (b) or CD1a+ (red) dendritic cells infiltrated in the cancer stroma (c). (d) Tumor‐infiltrating CD8+ T cells (red) are often Casitas–B‐lineage lymphoma protein‐b (Cbl‐b)+ (black). CD8+ T cells that have infiltrated and become attached to cancer cells (arrowhead) are Cbl‐b+ anergic cells. (e) FOXP3+ regulatory T cells (red) often express Cbl‐b (black). Arrowheads indicate regulatory T cells that have infiltrated and become attached to cancer cells. (a–e) Left column shows immunofluorescence staining for CD4 (a), CD8 (b, d), CD1a (c), or FOXP3 (e). Center column shows immunohistochemical staining for BTLA (a–c) or Cbl‐b (d, e). Right column shows merged images of left and center photographs.

Infiltration of T cell subsets, BTLA+ cells, and Cbl‐b+ cells

The median area of the ROI was 10 129 950 μm2 among the 212 cases of GBC, 1 336 266 μm2 among the 21 cases of CC, and 4 922 487 μm2 among the 11 cases of XGC. Representative photos of each type of positive cell are shown in Figure 2. Although there were too few samples of normal gallbladder tissue to compare with cancer tissue statistically, we found several CD3+ cells, CD4+ cells, and CD8+ cells in lamina propria. And a few FOXP3+ cells or Cbl‐b+ cells but no BTLA+ cells were found. Figure 3(a) shows a comparison of T cell subsets, BTLA‐positive cells, and Cbl‐b‐positive cells infiltrating into CC (n = 21), XGC (n = 11), and GBC (n = 211). The density of CD3+ cells, CD4+ cells, and CD8+ cells in CC and XGC was significantly higher than that in GBC, respectively (P < 0.01). In contrast, the density of FOXP3+ cells, BTLA+ cells, and Cbl‐b+ cells did not differ significantly among the diseases, except for the density of BTLA+ cells between CC and GBC. The number of suppressor cells such as regulatory T cells (Tregs) is often affected by the entire T cell infiltration.16, 18 It is often better to estimate the immune microenvironment from the ratio of the number of tumor‐infiltrating suppressor cells to that of the immune responsive cell population, rather than the absolute number of tumor‐infiltrating suppressor cells.16, 18, 42 We used the ratio of FOXP3 density to CD4 density (FOXP3/CD4), the ratio of BTLA density to CD8 density (BTLA/CD8), and the ratio of Cbl‐b density to CD8 density (Cbl‐b/CD8); comparisons of these ratios between the diseases are depicted in Figure 3(b). FOXP3/CD4, BTLA/CD8, and Cbl‐b/CD8 in GBC were significantly higher than those in CC and XGC, respectively (P < 0.01). Comparisons of the BTLA/CD3 and Cbl‐b/CD3 ratios among GBC, CC, and XGC showed similar profiles to those of BTLA/CD8 and Cbl‐b/CD8, respectively (Fig. S1).

Figure 2

Immunohistochemical features of tumor‐infiltrating cells. These representative photos show: (a) CD3+ T cells, (b) CD4+ T cells, (c) CD8+ T cells, (d) FOXP3+ T cells, (e) B and T lymphocyte attenuator (BTLA)+ cells, and (f) Casitas–B‐lineage lymphoma protein‐b (Cbl‐b+) cells. Scale bar = 100 μm. High power views are shown in insets of (e) and (f).

Figure 3

Comparison of cells infiltrating into chronic cholecystitis (CC, n = 21), xanthogranulomatous cholecystitis (XGC, n = 11), and gallbladder cancer (GBC, n = 211). (a) Density of immunolabeled cells (cells/μm2). (b) Density ratio of FOXP3+ cells to CD4+ T cells (FOXP3/CD4), that of B and T lymphocyte attenuator (BTLA)+ cells to CD8+ T cells (BTLA/CD8), that of BLTA + cells to CD4+ T cells (BTLA/CD4), that of Casitas–B‐lineage lymphoma protein‐b (Cbl‐b)+ cells to CD8+ T cells (Cbl‐b/CD8), and that of Cbl‐b+ cells to CD4+ T cells (Cbl‐b/CD4). The line in the middle of the boxes shows the median value. The bottom and top of the box indicates the 25th and 75th percentiles, respectively. The T‐bars that extend from the boxes show inner fences and “x” indicates outliers. Significant values were *P < 0.05 and **P < 0.01.

Interrelationships between clinicopathological variables and tumor‐infiltrating cells

We analyzed the interrelationships between clinicopathological variables of GBC and tumor‐infiltrating T cell subsets, BTLA+ cells, and Cbl‐b+ cells (Table 1). FOXP3/CD4, BTLA/CD8, and Cbl‐b/CD8 showed significant and positive correlations with each other. FOXP3/CD4 was significantly correlated with most of the conventional clinicopathological variables: tumor main location, T factor, N factor, M factor, histopathological grading, venous invasion, and perineural invasion. Likewise, BTLA/CD8 was significantly correlated with T factor, M factor, histopathological grading, venous invasion, and perineural invasion, and Cbl‐b/CD8 was significantly correlated with T factor and M factor. The interrelationships of BTLA/CD3 and Cbl‐b/CD3 with various factors were similar to those of BTLA/CD8 and Cbl‐b/CD8, respectively (data not shown).

Table 1

Interrelationships between clinicopathological variables and tumor‐infiltrating cells

Variables	CD3			CD4			CD8			FOXP3			FOXP3/CD4			BTLA			BTLA/CD8			BTLA/CD4			Cbl‐b			Cbl‐b/CD8			Cbl‐b/CD4
	Low	High	P‐value	Low	High	P‐value	Low	High	P‐value	Low	High	P‐value	Low	High	P‐value	Low	High	P‐value	Low	High	P‐value	Low	High	P‐value	Low	High	P‐value	Low	High	P‐value	Low	High	P‐value
Age, years			0.302			0.033			0.063			0.016			0.837			0.046			0.242			0.046			0.630			0.302			0.535
<68 (n = 105)	56	49		60	45		59	46		61	44		53	52		45	60		48	57		45	60		54	51		49	56		50	55
≥68 (n = 106)	49	57		45	61		46	60		44	62		52	54		60	46		57	49		60	46		51	55		57	49		55	51
Gender			0.046			0.948			0.833			0.002			0.537			0.948			0.627			0.733			0.046			0.627			0.085
Female (n = 100)	57	43		50	50		49	51		61	39		52	48		50	50		48	52		51	49		57	43		52	48		56	44
Male (n = 111)	48	63		55	56		56	55		44	67		53	58		55	56		57	54		54	57		48	63		54	57		49	62
Tumor main location			0.308			0.009			0.422			0.681			0.025			0.158			0.617			0.845			0.422			0.158			0.025
Gf, Gb (n = 104, 46)	78	72		66	84		72	78		76	74		82	68		70	80		73	77		74	76		72	78		80	70		82	68
Gn, C (n = 40, 21)	27	34		39	22		33	28		29	32		23	38		35	26		32	29		31	30		33	28		26	35		23	38
T			0.538			0.295			0.060			0.951			0.023			0.023			<0.001			0.005			0.372			0.003			0.011
1, 2 (n = 28, 66)	49	45		43	51		40	54		47	47		55	39		55	39		62	32		57	37		50	44		58	36		56	38
3, 4 (n = 74, 43)	56	61		62	55		65	52		58	59		50	67		50	67		43	74		48	69		55	62		48	69		49	68
N			0.836			0.448			0.448			0.063			0.033			0.007			0.113			0.301			0.113			0.946			0.190
0 (n = 107)	54	53		56	51		56	51		60	47		61	46		63	44		59	48		57	50		59	48		54	53		58	49
1 (n = 104)	51	53		49	55		49	55		45	59		44	60		42	62		46	58		48	56		46	58		52	52		47	57
M			0.963			0.522			0.126			0.398			0.009			0.083			0.003			0.020			0.802			0.034			0.009
0 (n = 145)	72	73		70	75		67	78		75	70		81	64		78	67		82	63		80	65		73	72		80	65		81	64
1 (n = 66)	33	33		35	31		38	28		30	36		24	42		27	39		23	43		25	41		32	34		26	40		24	42
Histopathological grading			0.303			0.239			0.727			0.063			0.003			0.063			0.033			0.033			0.632			0.146			0.191
1 (n = 99)	53	46		45	54		48	51		56	43		60	39		56	43		57	42		57	42		51	48		55	44		54	45
2, 3, 4 (n = 62, 45, 5)	52	60		60	52		57	55		49	63		45	67		49	63		48	64		48	64		54	58		51	61		51	61
Lymphatic invasion			0.016			0.352			0.962			0.069			0.128			0.035			0.069			0.128			0.128			0.403			0.128
0 (n = 68)	42	26		37	31		34	34		40	28		39	29		41	27		40	28		39	29		39	29		37	31		39	29
1 (n = 143)	63	80		68	75		71	72		65	78		66	77		64	79		65	78		66	77		66	77		69	74		66	77
Venous invasion			0.010			0.537			0.436			0.083			0.022			0.083			0.022			0.146			0.146			0.058			0.022
0 (n = 88)	53	35		46	42		41	47		50	38		52	36		50	38		52	36		49	39		49	39		51	37		52	36
1 (n = 123)	52	71		59	64		64	59		55	68		53	70		55	68		53	70		56	67		56	67		55	68		53	70
Perineural invasion			0.148			0.442			0.295			0.372			0.002			0.735			0.045			0.372			0.951			0.110			0.023
0 (n = 94)	52	42		44	50		43	51		50	44		58	36		48	46		54	40		50	44		47	47		53	41		55	39
1 (n = 117	53	64		61	56		62	55		55	62		47	70		57	60		51	66		55	62		58	59		53	64		50	67
CD3						<0.001			<0.001			<0.001			0.005			0.063			0.046			0.002			0.063			0.302			0.046
Low (n = 105)				77	28		74	31		71	34		42	63		59	46		45	60		41	64		59	46		49	56		45	60
High (n = 106)				28	78		31	75		34	72		63	43		46	60		60	45		64	42		46	60		57	49		60	46
CD4									<0.001			<0.001			<0.001			0.007			0.011			<0.001			<0.001			0.003			<0.001
Low (n = 105)							81	24		70	35		29	76		62	43		43	62		35	70		67	38		42	63		37	68
High (n = 106)							24	82		35	71		76	30		43	63		62	44		70	36		38	68		64	42		68	38
CD8												<0.001			<0.001			0.630			<0.001			<0.001			0.016			<0.001			0.001
Low (n = 105)										71	34		37	68		54	51		29	76		37	68		61	44		32	73		40	65
High (n = 106)										34	72		68	38		51	55		76	30		68	38		44	62		74	32		65	41
FOXP3															0.001			<0.001			0.945			0.837			0.001			0.449			0.837
Low (n = 105)													64	41		68	37		52	53		53	52		64	41		50	55		53	52
High (n = 106)													41	65		37	69		53	53		52	54		41	65		56	50		52	54
FOXP3/CD4																		0.063			<0.001			<0.001			0.837			<0.001			<0.001
Low (n = 105)																59	46		69	36		75	30		53	52		68	37		74	31
High (n = 106)																46	60		36	70		30	76		52	54		39	67		31	75
BTLA																					<0.001			<0.001			<0.001			<0.001			0.016
Low (n = 105)																			80	25		78	27		69	36		67	38		61	44
High (n = 106)																			25	81		27	79		36	70		39	67		44	62
BTLA/CD8																								<0.001			0.007			<0.001			<0.001
Low (n = 105)																						88	17		62	43		80	25		70	35
High (n = 106)																						17	89		43	63		26	80		35	71
BTLA/CD4																											0.033			<0.001			<0.001
Low (n = 105)																									60	45		73	32		73	32
High (n = 106)																									45	61		33	73		32	74
Cbl‐b																														<0.001			<0.001
Low (n = 105)																												76	29		75	30
High (n = 106)																												30	76		30	76
Cbl‐b/CD8																																	<0.001
Low (n = 105)																															90	16
High (n = 106)																															15	90

Density ratios are shown of FOXP3 to CD4 (FOXP3/CD4), B and T lymphocyte attenuator (BTLA) to CD8 (BTLA/CD8), BTLA to CD4 (BTLA/CD4), Casitas–B‐lineage lymphoma protein‐b (Cbl‐b) to CD8 (Cbl‐b/CD8), and Cbl‐b to CD4 (Cbl‐b/CD4). Bold values, P < 0.05; underlined values, positively correlated. C, cystic duct; Gb, gallbladder body; Gf, gallbladder fundus; Gn, gallbladder neck.

Prognostic significance of tumor‐infiltrating T cell subsets, BTLA+ cells, and Cbl‐b+ cells

Kaplan–Meier survival analyses revealed that a lower density of tumor‐infiltrating CD8+ T cells, and higher FOXP3/CD4, BTLA/CD8, and Cbl‐b/CD8 ratios were significantly associated with both shorter OS and DFS in GBC patients (Fig. 4). Higher BTLA/CD3 and Cbl‐b/CD3 ratios were closely associated with both shorter OS and DFS (Fig. S2). Five‐year survival rate, median survival time, and Cox analyses in the groups categorized by each of the parameters of tumor‐infiltrating cells and the conventional clinicopathological variables are summarized in Tables 2 and S1. When the variables that had been found to be significant by univariate analysis were subjected to multivariate analysis, M factor, perineural invasion, BTLA/CD8, and Cbl‐b/CD8 were closely associated with shorter OS. In addition, T factor, M factor, perineural invasion, and Cbl‐b/CD8 were significantly associated with shorter DFS.

Figure 4

Kaplan–Meier survival curves comparing (a) overall survival and (b) disease‐free survival in gallbladder cancer patients between the high (red) and low (blue) value groups with regard to the density of tumor‐infiltrating CD3+ T cells, CD4+ T cells, CD8+ T cells and the density ratio of the FOXP3+ T cells to CD4+ T cells (Foxp3/CD4), that of B and T lymphocyte attenuator (BTLA)+ cells to CD8+ T cells (BTLA/CD8), that of BTLA + cells to CD4+ T cells (BTLA/CD4), that of Casitas–B‐lineage lymphoma protein‐b (Cbl‐b)+ cells to CD8+ T cells (Cbl‐b/CD8), and that of Cbl‐b+ cells to CD4+ T cells (Cbl‐b/CD4). P‐values were obtained by log–rank test.

Table 2

Univariate and multivariate analyses for variables associated with overall survival (OS) and disease‐free survival (DFS) in patients with gallbladder cancer

Variables	Category	n	5‐year survival, %	Median survival time, months	OS								DFS
					Univariate				Multivariate				Univariate				Multivariate
					HR	95%CI		P‐value	HR	95%CI		P‐value	HR	95%CI		P‐value	HR	95%CI		P‐value
Age ≥68 years	No/yes	105/106	40.8/44.5	34.1/38.2	0.958	0.669	1.371	0.813					1.024	0.679	1.545	0.910
Gender	F/M	100/111	47.6/38.1	56.1/27.8	1.308	0.911	1.876	0.145					1.395	0.915	2.127	0.122
Location	Gf,Gb/Gn,C	104,46/40,21	48.4/29.6	54.6/27.1	1.422	0.973	2.078	0.069					1.667	1.092	2.543	0.018
T	1,2/3,4	28,66/74,43	71.5/19.2	215.2/17.8	4.587	3.013	6.986	<0.001					7.453	4.255	13.055	<0.001	2.906	1.431	5.898	0.003
N	0/1	107/104	58.3/25.8	215.2/19.5	2.713	1.870	3.937	<0.001					3.868	2.467	6.064	<0.001
M	0/1	145/66	54.7/15.1	138.8/12.9	3.772	2.607	5.457	<0.001	2.156	1.462	3.179	<0.001	3.922	2.562	6.004	<0.001	2.318	1.493	3.598	<0.001
Grading	1/2,3,4	99/62,45,5	61.5/26.1	215.2/19.5	3.071	2.049	4.535	<0.001					3.659	2.303	5.814	<0.001
L	0/1	68/143	71.7/27.9	215.2/22.9	3.500	2.195	5.581	<0.001					5.737	3.042	10.818	<0.001
V	0/1	88/123	69.4/22.3	215.2/19.8	3.861	2.505	5.814	<0.001					4.984	2.956	8.404	<0.001
Pn	0/1	94/117	71.7/18.7	215.2/17.2	4.715	3.093	7.187	<0.001	3.833	2.453	5.989	<0.001	7.758	4.424	13.605	<0.001	3.325	1.631	6.779	0.001
CD3	Low/high	105/106	41.5/43.7	29.4/43.6	0.824	0.576	1.179	0.291					1.152	0.762	1.742	0.501
CD4	Low/high	105/106	39.3/46.0	29.7/46.4	0.721	0.503	1.033	0.075					0.853	0.565	1.287	0.448
CD8	Low/high	105/106	30.0/56.6	22.9/191.0	0.478	0.331	0.691	<0.001					0.616	0.406	0.933	0.022
Foxp3/CD4	Low/high	105/106	61.1/26.8	NR/20.8	2.783	1.894	4.088	<0.001					1.855	1.217	2.826	0.004
BTLA/CD8	Low/high	105/106	65.8/20.0	191.0/20.8	3.577	2.387	5.359	<0.001	2.154	1.400	3.313	<0.001	2.757	1.766	4.302	<0.001
BTLA/CD4	Low/high	105/106	63.0/25.2	NR/24.6	2.892	1.957	4.274	<0.001					2.362	1.530	3.648	<0.001
Cbl‐b/CD8	Low/high	105/106	65.6/18.8	191.2/21.6	3.282	2.213	4.868	<0.001	2.374	1.565	3.602	<0.001	2.759	1.766	4.309	<0.001	2.445	1.555	3.847	<0.001
Cbl‐b/CD4	Low/high	105/106	65.2/22.5	NR/21.2	3.080	2.088	4.544	<0.001					2.675	1.723	4.153	<0.001

Density ratios are shown of FOXP3 to CD4 (FOXP3/CD4), B and T lymphocyte attenuator (BTLA) to CD8 (BTLA/CD8), BTLA to CD4 (BTLA/CD4), Casitas–B‐lineage lymphoma protein‐b (Cbl‐b) to CD8 (Cbl‐b/CD8), and Cbl‐b to CD4 (Cbl‐b/CD4). C, cystic duct; CI, confidence interval; F, female; Gb, gallbladder body; Gf, gallbladder fundus; Gn, gallbladder neck; HR, hazard ratio; L, lymphatic invasion; M, male; NR, not reached; Pn, perineural invasion; V, venous invasion. Bold values, P < 0.05.

Discussion

Here we evaluated for the first time the clinical impact of BTLA in cancer tissues. First, we assessed whether tumor‐infiltrating immune cells reflected the character of the tumor immune microenvironment, as it has been reported in various other cancers. The present study, including 211 cases of GBC, revealed that greater infiltration of CD8+ T cells and CD4+ T cells in the cancer tissue was a favorable prognostic indicator, as has been reported for many other cancers and for GBC.20, 21 It also showed that a higher prevalence of Tregs (FOXP3/CD4) was significantly correlated with malignant phenotypes and an unfavorable outcome, suggesting that Tregs play a role in controlling the immune response to GBC. Furthermore, a higher ratio of Cbl‐b+ anergic lymphocytes to CD8+ T cells (or to total T cells) was significantly correlated with advanced T factor, distant metastasis, and poorer prognosis. Therefore, the importance of antitumor immunity, represented by tumor‐infiltrating immune cells, in the outcome and control of cancer was confirmed for GBC.

Tumor cells are able to evade immune recognition and destruction through a variety of mechanisms. One such mechanism is the immunosuppressive action of co‐inhibitory molecules.23 BTLA is a co‐inhibitory molecule whose expression has been reported in mice and human blood cells. However, no previous study has investigated the expression of BTLA in the microenvironment of human tumors. Here, we found that BTLA was expressed on a proportion of several different types of tumor‐infiltrating immune cells, including CD3+ T cells, CD4+ T cells, CD8+ T cells, CD20+ B cells, CD14+ monocytes, CD68+ macrophages, CD1a+ DCs, CD207+ DCs, and CD208+ DCs. Our clinicopathological investigation revealed that BTLA/CD8 was increased in malignant disease (GBC) relative to benign disease (CC and XGC) and that the BTLA/CD8 ratio in tumor‐infiltrating immune cells was enhanced in patients with more advanced cancer. Furthermore, a higher BTLA/CD8 ratio was significantly associated with shorter DFS and OS. These findings suggest that BTLA is involved in the formation of a protumor microenvironment, supporting the contention that BTLA is an important co‐inhibitory molecule in the orchestration of immunosuppressive networks in cancer, and therefore a potential new target for interventions aimed at reversal of immune evasion and boosting of antitumor immunity in cancer patients.

It has been shown that BLTA binds to the herpes virus entry mediator (HVEM),43 inhibits T‐cell proliferation and cytokine production in vitro, and mediates the negative regulation of CD8+ T‐cell homeostasis and memory cell generation in vivo.44 In the context of cancer immunology, BTLA–HVEM is known to be another inhibitory pathway used by cancer cells to impair the antitumor immune response.45, 46, 47 HVEM is constitutively expressed on naive T cells and downregulated following T cell activation, only to be re‐expressed later on effector and memory T cells.48 It is also broadly expressed on cells of the immune system such as Tregs, B cells, monocytes, neutrophils, NK cells, and DCs, in addition to epithelial cells.49, 50 Although it would have been informative to examine the distribution of HVEM immunohistochemically, we were unable to do so, because of the lack of reliable antibody specific for HVEM. In contrast to the CTLA‐4 and PD‐1 pathways, HVEM binds to several receptor molecules, generating immune‐positive and ‐negative signals according to the receptors involved. Therefore, the total expression of BTLA in the tumor microenvironment might be a simpler and more accurate indicator of the protumor microenvironment, rather than expression of HVEM. It remains to be investigated whether a specific cell type with high BTLA expression exerts a predominantly inhibitory action or whether various types of immune cells function in a coordinated manner.

Maintenance of tolerance and induction of T cell anergy is critical for prevention of autoimmunity. However, in cases of malignancy, tumor‐induced T cell anergy and/or tolerance induces cancer‐associated immune paralysis, which contributes, at least in part, to uncontrolled tumor growth and metastasis. No previous studies have, in fact, demonstrated anergy cells in tissues. In the present study, we observed that T cells that had infiltrated and become attached to cancer cells were often positive for Cbl‐b, and sometimes positive for BTLA. Although no BTLA+Cbl‐b+ cells were found, T cells that had infiltrated within cancer nests were suggested to have become anergic under the influence of BTLA signals. Our clinicopathological analyses revealed that Cbl‐b/CD8 was increased in GBC relative to CC and XGC, and that Cbl‐b/CD8 in tumor‐infiltrating immune cells was higher in patients with more advanced cancer, as was the case for BTLA expression. Moreover, Cbl‐b/CD8 was significantly and positively correlated with BTLA/CD8 and FOXP3/CD4, and a higher Cbl‐b/CD8 ratio was an independent indicator of poor prognosis in terms of DFS and OS for patients with GBC. Accordingly, it is suggested that T cell anergy in the tumor microenvironment, reflected by the expression of Cbl‐b, represents promotion of a protumor microenvironment in GBC, in concert with BTLA. In addition, Cbl‐b expression may be a useful marker for evaluating T cell anergy in the tumor microenvironment. Here we have shown for the first time the usefulness of Cbl‐b for evaluation of the tumor microenvironment.

Therapeutic blockade of the immune checkpoint pathways, CTLA‐4 and PD‐1/PD‐L1, has shown promise in a variety of malignancies,23, 51 although the effectiveness of the reagents used, especially anti‐CTLA4 antibody, has been rather limited for major epithelial cancers in comparison with melanoma, and even in melanoma cases showing basic or acquired resistance have often been observed. In addition, a wide range of immune‐related adverse events have also been observed following treatment.23, 51 To increase the antitumor effect and to reduce the incidence of immune‐related adverse events, optimization of the dose and schedule of reagents, and characterization of biomarkers associated with disease outcome, have been extensively investigated. In addition, various combination approaches have been considered, including blockade of immune checkpoints together with the use of other anticancer treatments such as chemotherapy, radiotherapy, targeted therapy, and other forms of immunotherapy. Some combination approaches have achieved enhanced antitumor effects in animal models and human clinical studies,29, 30, 31 in which monotherapy has exerted only modest effects. Novel immune checkpoints are currently being investigated based on experience with CTLA‐4 and PD‐1/PD‐L1. Various combination approaches using these novel checkpoints may yield a therapeutic edge in the battle against a range of cancers. Therefore, it is suggested that our data for BTLA in GBC will be useful for future studies aimed at developing this new approach.

In conclusion, our findings suggest that BTLA in the tumor microenvironment plays an inhibitory role in the immune reaction against GBC, in concert with other factors. Higher expression of BTLA and Cbl‐b in tumor‐infiltrating immune cells appears to be an indicator of poor prognosis, whereas a higher ratio of non‐anergic CD8+ T cells is an independent favorable prognostic factor in GBC patients. Targeting BTLA for reversal of immune evasion may represent a promising new therapeutic approach.

Disclosure Statement

The authors have no conflict of interest.

Fig. S1. Comparison of infiltrating cells in gallbladder lesion.

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Fig. S2. Kaplan–Meier survival curves.

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Table S1. Univariate and multivariate survival analyses.

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