Characterization of tumor-associated B-cell subsets in patients with colorectal cancer

Abstract

Purpose: A precise understanding of the mechanisms by which human immune cell subsets affect tumor biology will be critical for successful treatment of cancer using immunotherapeutic approaches. Recent evidence suggests that B cells can both promote and inhibit the development and progression of tumors. The aim of this study was to characterize the composition of the B-cell infiltrates in colorectal cancers (CRC) in order to gain further insight into the role of B cells in CRC.

Experimental design: In this study we characterized B-cell subsets in primary tumors (n=38), metastases (n=6) and blood (n=46) of 51 patients with a diagnosis of CRC and blood of 10 healthy controls. B-cell subsets were analyzed by flow cytometry or immunohistochemistry.

Results: Peripheral blood of CRC patients contained a higher percentage of memory B cells than that of age-matched healthy controls. Furthermore, the percentage of B cells within tumors was higher than that in the peripheral blood of CRC patients while metastases were typically devoid of tumor-infiltrating B cells. Tumor-associated B cells were enriched for activated and terminally differentiated B cells. Relevant proportions of regulatory B cells could only be detected in advanced cancer and metastases.

Conclusion: B cells constitute a significant proportion of the immune infiltrate in CRC. The B-cell infiltrate of primary CRC is characterized by an accumulation of terminally differentiated memory B cells or plasma cells suggestive of a specific immune response against the tumor. However advanced tumors and metastases are also infiltrated by a considerable number of regulatory B cells.

Figure 1. B cells and B-cell subsets in PBMC and tumor samples of CRC patients and PBMC of healthy controls

(A) Flow cytometric analyzes of CD19⁺ cells in PBMCs of healthy controls (n=10), PBMCs of CRC patients (n=46) and single cell suspensions of tumor samples (n=38). The percentage of CD19⁺ cells in tumor samples is significantly higher than in PBMC of CRC patients (8.9% vs. 5.1% of CD45+ lymphocytic cells, p<0,05). (B) Tumor associated B cells (n=28) contain a higher percentage of activated B cells (defined by positivity for CD19, CD20 and CD86) than PBMCs of CRC patients (n=44) or healthy controls (n=10) (16.8% vs. 4.1/4.5% respectively, p<0,005). (C) CD27⁺IgD⁻ (memory) B cells are elevated in PBMCs (n=44) and tumor (n=28) of colorectal cancer patients compared to PBMCs of healthy controls (n=10) (19.3/22.7% vs. 8.2%, p<0,05/<0,005 respectively). (D) According to an increase in memory B cells virgin naïve (IgD+/CD27-/CD38-) B cells are decreased in PBMCs (n=44) and tumor samples (n=28) of CRC patients compared to PBMC of healthy controls (n=10) (p<0,05) (E) The percentage of IgD+/CD27-/CD38- naive B cells decreases with UICC stage (n=28, n.s.). (F) Exemplary immunohistochemical staining of CD20 to visualize the intratumoral distribution of tumor-associated B cells, predominantly in peritumorous lymphoid follicles. (G) Immunohistochemical analyzes of Ki-67 in CD20⁺ cells revealed a strong local proliferation of B cells within tertiary lympoid structures.

Figure 2. Plasma cells in PBMC and tumor samples of CRC patients and PBMC of healthy controls

(A) Plasma cells (CD19⁺CD20⁻CD38^high) are strongly elevated in PBMCs (n=46) and tumor tissue (n=38) of CRC patients compared to healthy controls (n=10) (12.6%/31.7% vs. 1.4% respectively; p<0,05/<0,005) (B) Immunohistochemistry showing an infiltration of colorectal cancer by IgG-kappa⁺ cells into the same tumor sample as shown in C. (C) Exemplary density plots showing CD45⁺CD19⁺CD20⁻CD38^high cells in peripheral blood and tumor tissue of CRC patients and peripheral blood of healthy controls. The plasma cell phenotype of these cells was confirmed by expression of CD27.

Figure 3. Regulatory B-cell subsets in Colorectal Cancer

(A) The percentage of CD24^highCD38^high (transitional) B cells is lower in tumor samples (n=28) than in peripheral blood of CRC patients (n=44) or healthy controls (n=10) (1.2% vs. 3.5%/2,3%, p<0,005/p<0,05 respectively). (B) Whereas transitional B cells are very rare in early stage CRC the frequency increases in advanced stage disease (n=28) (0.7% of B cells in Stage I+II vs. 1.8% in Stage III+IV, p<0,05). (C) CD24^highCD27⁺ B cells make up a high percentage of tumor associated B cells in tumor samples (n=28) and PBMC of colorectal cancer (n=44) and are significantly elevated compared to PBMC of healthy controls (n=10) (21.1%/18.5% vs. 11.3%, p<0,05). (D) Exemplary density plot showing CD24^highCD27⁺ B cells in a tumor sample from a patient with colorectal cancer (gated on lymphocytic FSC/SSC,CD45⁺,CD19⁺,CD20⁺).

Figure 4. Memory B cells and cells with a plasma cell phenotype are significantly reduced in colorectal liver metastasis

(A) CD19⁺CD20⁺ cells make up only 2% of CD45+ lymphocytic cells in metastatic tissue (n=6) compared to 6.9% in samples of primary colorectal cancer (n=28) (p<0,05) (B) The percentage of IgD-/CD27+ memory b cells in colorectal liver metastasis (n=6) is significantly lower than in primary tumors (n=28) (9.9% vs. 23.4%, p<0,05) (C) Whereas cells with a plasma cell phenotype were regularly detectable in tumor samples of CRC patients (n=38) they were heavily reduced in metastatic Tissue (n=6) (3.7% compared to 31.7%, p<0,005). (D) B cells with a regulatory phenotype (CD24^highCD38^high B cells) were rare in the primary tumors, but well detectable in metastatic tissue (1,1% vs. 6,3% of CD19⁺CD20⁺ cells, p<0,05).

Figure 5. Tumor infiltrating T cells in colorectal cancer

(A) Tumor infiltrating T cells are mainly of a CD45RA⁻CCR7⁻ effector/memory (EM) phenotype (n=37) whereas PBMC of CRC patients (n=38) and healthy controls (n=10) contain predominantly naive (CD45RA⁺CCR7⁺) and central memory (CM) (CD45RA⁻CCR7⁺) T cells. (B) The percentage of CD45RA⁻CCR7⁻ effector memory T cells in primary tumor samples is significantly reduced in Stage IV colorectal cancer (65% vs. 77,9%, p<0,05) (C) CD3⁺CD4⁺CD25⁺CD127^low T cells which are known to contain a high percentage of FoxP3+ regulatory T cells are significantly elevated in tumor samples (n=38) compared to PBMC of CRC patients (n=39) and healthy controls (n=10) (15.5% of CD4⁺-Tcells in tumor samples compared to 7.8% and 5.0% respectively, p<0,005) (D,E) Immunohistochemical staining of CD20 and CD8 were performed to illustrate the intratumoral distribution of B and T cells. B cells were detectable throughout the whole tumor samples, but were predominantly localized in peritumoral tertiary lymphoid structures. Figure E highlights the infiltration of Colorectal Cancer by cytotoxic T cells and also demonstrates a colocalisation of cytotoxic T and B cells especially within the tertiary lymphoid structures. Sequential immunohistochemistries of CD8⁺-T cells and CD20⁺-B cells revealed similar colocalisations in 40% of the analyzed samples.