PD-L1 mediated the differentiation of tumor-infiltrating CD19⁺ B lymphocytes and T cells in Invasive breast cancer

Figures & data

Figure 1. Immunochistochemical staining of CD19 and PD-L1 in IBCa and fibroadenoma tissue of breast. Sections of fibroadenoma tissues and IBCa were stained with anti-CD19, anti-PD-1 and anti-PD-L1 antibodies, as described in “Materials and Methods”, shown in Fig. 1 are representative microphotographs (× 400). (A) The density of CD19⁺ B lymphocytes is significantly higher in IBCa tissue than that in fibroadenomas of breast. (B) The density of CD19⁺ B lymphocytes in grade 1, 2 and 3 IBCa. C: PD-L1 is strongly expressed in the cell membrane, cytoplasm or both the IBCa tissues than that in fibroadenomas of the breast. Isotype-matched mouse IgG1 mAb was used as control.

Figure 2. The colocalization of CD19 and IL-10 expression in IBCa tissues. The colocalization of CD19 and IL-10 expression in IBCa were analyzed by immunohistochemistry and immunoflurescence staining as described in “Materials and Methods”, shown in Fig. 2 are representative microphotographs (× 400). (A) The immunohistochemistry staining of serial sections showed the location of CD19 expression as well as IL-10 in TILs of IBCa tissue. (B) Confocal Laser-Scanning Microscopy was applied to detect CD19 and IL-10 in IBCa specimens. Membrane of B cells was visualized by CD19 staining (Green), cytoplasm are stained with IL-10 (Red), and nuclei are counterstained with DAPI (Blue). Merged figure exhibit a colocalization of CD19 and IL-10 in the B cells.

Table 1. Comparisons of the frequency of tumor-infiltrating B cells in breast fibroadenoma and IBCa tissue.

		CD19^+ B cells
	All case	–	1+	2+	3+	p
Fibroadenoma	31	23(74.2%)	5(16.1%)	3(9.7%)	0(0.0%)	<0.0001
IBCa	134	41(30.6%)	25(18.6%)	17(12.7%)	51(38.1%)

Table 2. Relationship between the tumor-infiltrating CD19⁺ B cell density and the clinicopathological parameters of IBCa patients.

		CD19^+ B cell
Variables	All cases	–	1+	2+	3+	p
IBCa	134	41(30.6%)	25(18.7%)	17(12.7%)	51(38.1%)
Grade
G1	52	22(42.3%)	10(19.2%)	7(13.5%)	13(25.0%)	<0.0001
G2	52	16(30.8%)	14(26.9%)	5(9.6%)	17(32.7%)
G3	30	3(10.0%)	1(3.3%)	5(16.7%)	21(70.0%)
LN metastasis
No	81	23(28.4%)	17(21.0%)	12(14.8%)	29(35.8%)	0.984
Yes	53	18(34.0%)	8(15.1%)	5(9.4%)	22(41.5%)
TNM
T1	40	11(27.5%)	4(10.0%)	7(17.5%)	18(45.0%)	0.438
T2	51	14(27.5%)	15(29.4%)	4(7.8%)	18(35.3%)
T3	31	12(38.7%)	6(19.4%)	4(12.9%)	9(29.0%)
T4	12	4(33.3%)	0(0.0%)	2(16.7%)	6(50.0%)
ER
Positive	86	28(32.6%)	19(22.1%)	14(16.3%)	25(29.1%)	0.047
Negative	48	13(27.1%)	6(12.5%)	3(6.3%)	26(54.2%)
PR
Positive	82	26(31.7%)	18(22.0%)	14(17.1%)	24(29.3%)	0.103
Negative	52	15(28.8%)	7(13.5%)	3(5.8%)	27(51.9%)
HER2
Positive	21	4(19.0%)	3(14.3%)	2(9.5%)	12(57.1%)	0.069
Negative	113	37(32.7%)	22(19.5%)	15(13.3%)	39(34.5%)
Age
<49 y	61	17(27.9%)	10(16.4%)	10(16.4%)	24(39.3%)	0.455
≥49 y	73	24(32.9%)	15(20.5%)	7(9.6%)	27(37.0%)
Tumor size
<3cm	74	15(20.3%)	16(21.6%)	11(14.9%)	32(43.2%)	0.021
≥3cm	60	26(43.3%)	9(15.0%)	6(10.0%)	19(31.7%)

Table 3. PD-L1 expression in breast fibroadenoma and IBCa tissue.

		PD-L1 expression
	All case	–	membrane +	cytoplasm +	both +	p
Fibroadenoma	31	9(29.0%)	3(9.7%)	2(6.5%)	17(54.8%)	0.001
IBCa	134	15(11.2%)	4(3.0%)	3(2.2%)	112(83.6%)

Table 4. Relationship between PD-L1 expression in IBCa tissue with histopathological features of IBCa.

		PD-L1 expresion
Variables	All cases	−	Membrane +	Cytoplasm +	Both^+	p
IBCa	134	15(11.2%)	4(3.0%)	3(2.2%)	112(83.6%)
Grade
G1	52	9 (17.3%)	2 (3.8%)	1 (1.9%)	40 (76.9%)*	0.063
G2	52	2 (3.8%)	1 (1.9%)	2 (3.8%)	47 (90.4%)
G3	30	4 (13.3%)	1(3.3%)	0 (0.0%)	25 (83.3%)
LN metastasis
No	81	10 (12.3%)	3 (3.7%)	3 (3.7%)	65 (80.2%)	0.359
Yes	53	5 (9.4%)	1 (1.9%)	0 (0.0%)	47 (88.7%)
TNM
T1	40	7 (17.5%)	3 (7.5%)	0 (0.0%)	30 (75.0%)	0.030
T2	51	6 (11.8%)	1 (2.0%)	2 (3.9%)	42 (82.3%)
T3	31	1 (3.2%)	0 (0.0%)	1 (3.2%)	29 (93.5%)
T4	12	1 (8.3%)	0 (0.0%)	0 (0.0%)	11 (91.7%)
ER
Positive	86	10 (11.6%)	1 (1.2%)	2 (2.3%)	73 (84.9%)	0.724
Negative	48	5 (10.4%)	3 (6.3%)	1 (2.1%)	39 (81.3%)
PR
Positive	82	9 (11.0%)	2 (2.4%)	1 (1.2%)	70 (85.4%)	0.685
Negative	52	6 (11.5%)	2 (3.8%)	2 (3.8%)	42 (80.8%)
HER2
Positive	21	0 (0.0%)	1 (4.8%)	0 (0.0%)	20 (95.2%)	0.104
Negative	113	15 (13.3%)	3 (2.7%)	3 (2.7%)	92 (81.4%)
Age
<49 y	61	6 (9.8%)	2 (3.3%)	1 (1.6%)	52 (85.2%)	0.663
≥49 y	73	9 (12.3%)	2 (2.7%)	2 (2.7%)	60 (82.2%)
Tumor size
<3cm	74	10 (13.5%)	4 (5.4%)	1 (1.4%)	59 (79.7%)	0.157
≥3cm	60	5 (8.3%)	0 (0.0%)	2 (3.3%)	53 (88.3%)

Table 5. Relationship between IL-10 expression in IBCa tissue with histopathological features of IBCa.

		IL-10 expression
Variables	All cases	–	+	p
IBCa	134	71 (53.0%)	63 (47.0%)
Grade
G1	52	31 (59.6%)	21(40.4%)	0.048
G2	52	30(57.7%)	22 (42.3%)	z=−2.114
G3	30	10 (33.3%)	20 (66.7%)
LN metastasis
No	81	40 (49.4%)	41(50.6%)	0.302
Yes	53	31 (58.5%)	22 (41.5%)
TNM
T1	40	23 (57.5%)	17(42.5%)	0.346
T2	51	22 (43.1%)	29 (56.9%)	z=0.380
T3	31	19 (61.3%)	12 (38.7%)
T4	12	7 (58.3%)	5 (41.7%)
ER
Positive	86	52 (60.5%)	34 (39.5%)	0.020
Negative	48	19 (39.6%)	29 (60.4%)
PR
Positive	82	49 (59.8%)	33 (40.2%)	0.049
Negative	52	22 (42.3%)	30 (57.7%)
HER2
Positive	21	7 (33.3%)	14 (66.7%)	0.049
Negative	113	64 (56.6%)	49 (43.4%)
Age
<49 y	61	34 (55.7%)	27 (44.3%)	0.559
≥49 y	73	37 (50.7%)	36 (49.3%)
Tumor size
<3 cm	74	39 (52.7%)	35 (47.3%)	0.942
≥3 cm	60	32 (53.3%)	28 (46.7%)

Table 6. Correlation of tumor-infiltrating CD19⁺ B cells with PD-L1 expression in cell membrane and cytoplasm of IBCa tissue.

	PD-L1 expression
	+	–	p and κ
CD19+ n	78	9	p = 0.001
CD19– n	34	6	κ=0.056

Table 7. Correlation of tumor-infiltrating CD19⁺ B cells with IL-10 expression in IBCa tissue.

	IL-10 expression
	+	–	p and κ
CD19+ n	52	41	p = 0.001
CD19– n	11	28	κ = 0.227

Figure 3. PD-L1 mediates the differentiation of CD19⁺ B cells into CD19⁺CD24⁺CD38⁺ B cell subtype. (A) The expression of PD-L1 and PD-1 on breast cancer cell lines was analyzed by flow cytometry. MDA-MB231 expresses high level of PD-L1 (94.7%) compared with MCF-7 (17.3%); Both MCF-7 and MDA-MB231 lacks PD-1 expression. (B) CD19⁺ B lymphocytes/ breast cancer cells co-culture systems were established to investigate the interaction between PD-L1 expression in breast cancer cells and CD19⁺ B cells. 16.1% of CD19⁺CD38⁺CD24⁺ B cells were detected in PD-L1^hi MDA-MB231, while only 9.38% of CD19⁺CD38⁺CD24⁺ B cells were detected in PD-L1^lo MCF-7. (C) To confirmed the relationship between PD-L1 and CD19⁺ B cells, PD-L1siRNA were used to knockdown the expression of PD-L1. (D) Co-culture of CD19⁺ B cells and PD-L1^hi MDA-MB231 with or without siRNA. The percentage of CD19⁺CD38⁺CD24⁺ B cells were reduced when CD19⁺ B cells were co-cultured with MDA-MB231 cells with PD-L1 knocked down. (E) A high level of IL10 was detected in PD-L1^hi co-culture compared to that in PD-L1^lo co-culture system (P < 0.05). And CD19⁺CD24⁺CD38⁺ B cells produced IL-10 following activation by PMA and ionomycin.

Figure 4. Phenotype and function of CD19⁺ B cells from IBCa patients. To investigate the phenotype and function of CD19⁺ B cells in patients and healthy individuals, CD19⁺ B cells were isolated from peripheral blood or breast tumor tissues, incubated with CD19-ECD, CD38-APC and CD24-PE-cy7, and analyzed by flow cytometry. CD4⁺ T cells in patients and healthy individuals were purified and incubated with CD4-ECD, CD25-PE-cy7, fixed and stained by Foxp3-APC. B/T lymphocytes co-culture systems were set up to examine the interaction between CD19⁺ B cells and CD4⁺ T cells. (A) Higher percentage of CD19⁺CD38⁺CD24⁺ B cells were detected in fresh IBCa tissues that in breast fibroadenoma tissue (6.38% vs. 2.79%). (B) Significantly higher level of CD19⁺CD38⁺CD24⁺ B cells was detected in the PBMC of IBCa patients compared with that in fibroadenoma or health individuals (p = 0.0176). (C) Elevated expression of IL-10 (36.4%) was detected in CD19⁺CD24⁺CD38⁺ B cells activated by LPS, PMA and ionomycin compared to that in CD19⁺CD24⁺CD38⁻ B cells (2.90%, P < 0.05). (D) A total of 80.4% of CD4⁺CD25⁺Foxp3⁺ T cells were found in the co-culture system, in which both CD19⁺ B cells and CD4⁺ T cells were originated from IBCa patients, versus 30.8% of CD4⁺CD25⁺Foxp3⁺ T cells in co-culture system of CD19⁺ B cells from patients and CD4⁺ T cells from health individuals. However, only 7.53% and 8.13% of CD4⁺CD25⁺Foxp3⁺ T cells were detected in the co-culture system of CD19⁺ B cells from health individuals with CD4⁺ T cells originated from health individual or IBCa patients, respectively. (E) Significantly higher level of CD4⁺CD25⁺Foxp3⁺ Treg cells were detected in the PBMC of IBCa patients compared with that in fibroadenoma patients or health individuals (P < 0.0001).

Figure 5. PD-L1 contributed to the induction of the CD4⁺CD25⁺Foxp3⁺ Tregs by CD19⁺ B cells in in vitro system. The potential role of PD-L1 in the B cells and T cells interaction were examined in co-culture experiments of CD4⁺ T cell with CD19⁺ B cells, or CD19⁺ B cells pre-cultured with MCF-7 or MD-MB231 cells, or MD-MB231 cells with or without PD-L1 knocked down. CD4⁺ T cells were isolated by CD4⁺ positive selection kit from peripheral blood of healthy individuals. Following activation with anti-human CD3 and anti-human CD28 antibodies, CD4⁺ T cells and B cells as described above were mixed together at a ratio of 1:1, and analyzed by flow cytometry following culture for the specified time as described in the Materials and Methods section. (A) A total of 48.6% CD4⁺CD25⁺ Foxp3⁺ T cells were produced in co-culture system in which B cells were pre-cultured (or stimulated) by PD-L1^hi MD-MB231, 21.8% CD4⁺CD25⁺ Foxp3⁺ T cells in co-culture system of T cells with B cells stimulated by PD-L1^lo MCF-7. (B) A total of 5.09% and 12.5% of CD4⁺CD25⁺ Foxp3⁺ T cells were detected in co-culture with B cells stimulated by MD-MB231 with or without PD-L1 depletion by PD-L1-siRNA or PD-L1-NC (negative control), respectively.