Epstein-Barr Virus+ B Cells in Breast Cancer Immune Response: A Case Report

Abstract

EBV-specific T cells have been recently described to be involved in fatal encephalitis and myocarditis in cancer patients after immune checkpoint therapies. Here, we report the study of a human triple-negative breast cancer tumor (TNBC) and EBV-transformed B cells obtained from a patient-derived xenograft (PDX) that progressed into a lymphocytic neoplasm named xenograft-associated B-cell lymphoma (XABCL). T-cell receptor (TCR) high-throughput sequencing was performed to monitor the T-cell clonotypes present in the different samples. Forty-three T-cell clonotypes were found infiltrating the XABCL tissue after three passes in mice along 6 months. Eighteen of these (42%) were also found in the TNBC biopsy. TCR infiltrating the XABCL tissue showed a very restricted T-cell repertoire as compared with the biopsy-infiltrating T cells. Consequently, T cells derived from the TNBC biopsy were expanded in the presence of the B-cell line obtained from the XABCL (XABCL-LCL), after which the TCR repertoire obtained was again very restricted, i.e., only certain clonotypes were selected by the B cells. A number of these TCRs had previously been reported as sequences involved in infection, cancer, and/or autoimmunity. We then analyzed the immunopeptidome from the XABCL-LCL, to identify putative B-cell-associated peptides that might have been expanding these T cells. The HLA class I and class II-associated peptides from XABCL-LCL were then compared with published repertoires from LCL of different HLA typing. Proteins from the antigen processing and presentation pathway remained significantly enriched in the XABCL-LCL repertoire. Interestingly, some class II-presented peptides were derived from cancer-related proteins. These results suggest that bystander tumor-infiltrating EBV+ B cells acting as APC may be able to interact with tumor-infiltrating T cells and influence the TCR repertoire in the tumor site.

Keywords: B cells; Epstein–Barr virus; T cells; TCR—T-cell receptor; breast cancer.

Figure 1

CD3+ T cells (both CD4+ and CD8+) and CD20+ B cells were present and colocalized in the patient biopsy. Hematoxylin and eosin (HE) staining and immunohistochemistry (IHC) from the triple-negative breast cancer (TNBC) core biopsy realized after diagnosis. (A) Histological section stain from breast carcinoma (HE ×200); (B) CD3 IHC stain in breast carcinoma highlighted numerous CD3+ T cells in the infiltrating front of neoplasia (×200); (C) CD20 IHC stain showed a similar distribution of CD20+ B cells compared with CD3+ T lymphocytes (×200); (D) CD4 IHC stain revealed that most lymphocytes are CD4+ T cells (×200); (E) CD8 IHC stain highlighted less CD8+ T cells but nearest to the tumor in the infiltrating front (×200).

Figure 2

The tumor derived from a TNBC patient implanted in a PDX was a B-cell lymphocytic tumor (XABCL). CD20 immunohistochemical stain in the histological section revealed that most of the cells were CD20+ B cells. Cells with a bilobed nucleus, reminding of the Reed–Sternberg cells associated with Hodgkin’s lymphoma, were observed (pointed with a red arrow).

Figure 3

Some T-cell receptor (TCR) sequences from the XABCL tissue were present in the TNBC tissue and the TNBC-T cells, but the three samples presented different TCR repertoire patterns. (A) UpSet plots represent the number of shared TCR sequences between samples. Eighteen TCR sequences were found in the XABCL and the TNBC biopsy and/or the TNBC-T cells. Twenty-five sequences were exclusively found in the XABCL. (B) The heat plot of the 50 most abundant clonotypes in the shared samples revealed a global different pattern of the TCR repertoire infiltrating the XABCL.

Figure 4

TNBC-T cells expanded in vitro in the presence of XABCL-LCL presented a highly restricted TCR repertoire with a different pattern. (A) The TCR repertoire size and diversity hugely decreased in the TNBC-T cell sample expanded in the presence of irradiated XABCL-LCL (60 Gy), compared with TNBC-T cells expanded with OKT3 (anti-human CD3). (B) The heat plot using the 50 most abundant clonotypes revealed that the TCR repertoire pattern of expanded TBNC-T cells was also different in the T cells grown with XABCL-LCL, instead of with OKT3, as several sequences were not found in common and different frequencies of the shared clonotypes were observed ( Supplementary Table 3 ).

Figure 5

TCR sequences from XABCL-infiltrating T cells and TNBC-T cells expanded in the presence of XABCL-LCL found in the McPAS-TCR database. Ten sequences from the 43 clonotypes (23%) infiltrating the XABCL tissue had been previously reported as TCR sequences recognizing pathogens. From these, 50% had also been described in non-communicable diseases, i.e., cancer and autoimmunity. One sequence had been identified in the three pathologies. From the T cells expanded in vitro in the presence of XABCL-LCL, 13 clonotypes were found in the literature: 8, 2, and 1 of these had been exclusively identified in pathogen infections, autoimmunity, and cancer, respectively, and 2 sequences had been described in the three categories, but remarkably the CASSIQETQYF sequence had been identified several times in cancer.

Figure 6

Distribution of the selected proteins obtained from the XABCLLCL HLA repertoire based on their expression in cancer. After comparing the source proteins with those found in the literature, the expression in cancer of 178 (HLA-ABC-associated) and 102 (HLA-DR-associated) proteins was checked by the Human Protein Atlas. Counts of proteins, peptides, and PSM obtained are represented. The highest count in all the measures corresponded to proteins with low cancer specificity, i.e., proteins enriched in many cancer tissues. Two proteins from HLA-ABC-derived peptides showed specificity in certain cancers. Regarding the HLA-DR results, peptides with high PSM values corresponded to specific proteins from thyroid cancer (280 PSM and 8 peptides contained in 1 nested set and derived from 1 protein), glioma (203 PSM and 6 peptides derived from 2 proteins), and liver cancer (146 PSM and 6 peptides derived from 3 proteins).