ORIGINAL RESEARCH published: 16 November 2021 doi: 10.3389/fimmu.2021.761798 Epstein–Barr Virus+ B Cells in Breast Cancer Immune Response: A Case Report Andrea Aran 1, Vicente Peg 2, Rosa Maria Rabanal 3, Cristina Bernadó 4, Esther Zamora 5, Elisa Molina 1, Yago A. Arribas 1, Joaquı́n Arribas 4,6,7,8, José Pérez 9, Carme Roura-Mir 1, Montserrat Carrascal 10, Javier Cortés 5,9 and Mercè Martı́ 1* 1 Immunology Unit, Department of Cell Biology, Physiology and Immunology, Institut de Biotecnologia i Biomedicina (IBB), Universitat Autònoma de Barcelona (UAB), Bellaterra, Spain, 2 Translational Molecular Pathology, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain, 3 Unitat de Patologia Murina i Comparada, Department of Animal Medicine and Surgery, Veterinary Faculty, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain, 4 Preclinical and Translational Research Program, Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain, 5 Breast Cancer Unit, Vall d’Hebron Institute of Oncology (VHIO), Hospital Universitari Vall d’Hebron, Barcelona, Spain, 6 Cancer Research Program, Hospital del Mar Medical Research Institute (IMIM), Barcelona, Spain, 7 Centro de Investigación Biomédica en Red de Cáncer, Madrid, Spain, 8 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain, 9 International Breast Cancer Center (BCC), Quironsalud Group, Barcelona, Spain, 10 Biological and Environmental Proteomics, Institute of Biomedical Research of Edited by: Barcelona, Spanish National Research Council, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IIBB-CSIC/ Zong Sheng Guo, IDIBAPS), Barcelona, Spain Roswell Park Comprehensive Cancer Center, United States EBV-specific T cells have been recently described to be involved in fatal encephalitis and Reviewed by: Bingliang Fang, myocarditis in cancer patients after immune checkpoint therapies. Here, we report the study University of Texas MD Anderson of a human triple-negative breast cancer tumor (TNBC) and EBV-transformed B cells Cancer Center, United States obtained from a patient-derived xenograft (PDX) that progressed into a lymphocytic Frank Momburg, German Cancer Research Center neoplasm named xenograft-associated B-cell lymphoma (XABCL). T-cell receptor (TCR) (DKFZ), Germany high-throughput sequencing was performed to monitor the T-cell clonotypes present in the *Correspondence: different samples. Forty-three T-cell clonotypes were found infiltrating the XABCL tissue after Mercè Martı´ merce.marti@uab.cat 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 Specialty section: compared with the biopsy-infiltrating T cells. Consequently, T cells derived from the TNBC This article was submitted to Cancer Immunity biopsy were expanded in the presence of the B-cell line obtained from the XABCL (XABCL- and Immunotherapy, LCL), after which the TCR repertoire obtained was again very restricted, i.e., only certain a section of the journal clonotypes were selected by the B cells. A number of these TCRs had previously been Frontiers in Immunology reported as sequences involved in infection, cancer, and/or autoimmunity. We then analyzed Received: 20 August 2021 Accepted: 21 October 2021 the immunopeptidome from the XABCL-LCL, to identify putative B-cell-associated peptides Published: 16 November 2021 that might have been expanding these T cells. The HLA class I and class II-associated Citation: peptides from XABCL-LCL were then compared with published repertoires from LCL of Aran A, Peg V, Rabanal RM, Bernadó C, Zamora E, Molina E, different HLA typing. Proteins from the antigen processing and presentation pathway Arribas YA, Arribas J, Pérez J, remained significantly enriched in the XABCL-LCL repertoire. Interestingly, some class II- Roura-Mir C, Carrascal M, Cortés J presented peptides were derived from cancer-related proteins. These results suggest that and Martı´ M (2021) Epstein–Barr Virus+ B Cells in Breast Cancer bystander tumor-infiltrating EBV+ B cells acting as APC may be able to interact with tumor- Immune Response: A Case Report. infiltrating T cells and influence the TCR repertoire in the tumor site. Front. Immunol. 12:761798. doi: 10.3389/fimmu.2021.761798 Keywords: Epstein–Barr virus, B cells, T cells, breast cancer, TCR—T-cell receptor Frontiers in Immunology | www.frontiersin.org 1 November 2021 | Volume 12 | Article 761798 Aran et al. EBV+ B Cells in Breast Cancer 1 INTRODUCTION EBV+ B cells in an infectious stage of the virus induce a strong T-cell-specific response (23). However, under the influence of the The immune system plays a key role in cancer, but differences tumor microenvironment and/or of immunomodulatory between individuals can lead to different outcomes. A high treatments, the behavior, response, and repertoire of tumor- number of tumor-infiltrating lymphocytes (TIL) in triple- infiltrating cells—not only tumor-specific T cells but also negative breast cancer (TNBC) are related to a better prognosis bystander EBV-specific T cells—can be affected. This would (1), although not in all patients (2). Several factors influence a give an opportunity for EBV in an infectious stage to survive good antitumoral immune response, such as the recognition of but also could have long-term consequences affecting the neoantigens, the presence of immunomodulatory mediators, or immune system. The presence of EBV-specific T cells among the cellular composition and diversity of TIL, including the tumor infiltrates has been described, and in two recent studies, presence of antigen-presenting cells (APC) such as dendritic their involvement in fatal inflammatory processes was suggested cells (DC) or B cells. DC can migrate from the tumor site to the [myocarditis (24) and encephalitis (25)] in melanoma patients proximal lymph nodes to activate antigen-specific T cells, but after immune checkpoint inhibitor (ICPI) therapies. the presence of APC in the tumor site is also necessary for the The aim of this study was to determine the relationship maintenance of cytotoxic T lymphocytes (CTL) and T helper between tumor-infiltrating T and EBV+ B cells from a TNBC (Th) cells. In the last years, tumor-associated B cells have been patient. To our knowledge, this is the first time that B cells gaining interest and their presence has been associated with a generated in a XABCL have been used as an autologous EBV good prognosis in breast cancer (3), colorectal cancer (4), ovarian lymphoblastoid cell line (LCL) to study these B-cell–TIL cancer (5), hepatocellular carcinoma (6), soft sarcoma (7), and interactions. T-cell receptor (TCR) and B-cell receptor (BCR) lung cancer (8), among others. Nevertheless, the role of B cells as analyses were performed to study the presence of tumoral- active APC in the tumor site is not well established. infiltrating lymphocytes in the TNBC tumor as well as to B cells are also the main targets of the Epstein–Barr virus monitor T-cell expansions. Our data as a first proof of concept (EBV) (9). Asymptomatic infections of young children by EBV have demonstrated that TIL derived from the breast cancer are very common, so it is not surprising that EBV-infected cells infiltrate can be maintained for at least 6 months in the (EBV+ B cells) are found among TIL (10) in many cancer types. XABCL, but also that certain clonotypes can be expanded EBV was the first recognized human oncovirus when it was in vitro in the presence of the established B-cell line derived isolated from a Burkitt lymphoma patient (9). Nowadays, the from the XABCL (XABCL-LCL). Some of the TCRs involved relationship between EBV infection and several lymphomas, i.e., have been previously described in cancer, but also in infections Burkitt lymphoma (9, 11), Hodgkin’s lymphoma (12, 13), and and autoimmunity. The whole peptide repertoire presented by posttransplant lymphoproliferative disorder (14, 15) is HLA-ABC and HLA-DR from XABCL-LCL was also well established. investigated and compared with LCL peptidomes from the In the last years, the influence of endogenous virus in the literature. The characterization of the specific proteins from outcome of carcinomas has been gaining evidence (16). which these peptides were derived indicated that most of them Nevertheless, most of the studies are focused on the role of were related with antigen processing and presentation pathways, these viruses in the malignant transformation of epithelial cells, suggesting that the XABCL-LCL can act as a strong APC. We but the influence of virus-infected immune cells in the have also assessed whether some of the proteins identified in the antitumoral response should also be considered. Even though immunopeptidome had a relationship with cancer, and epithelial cells can also be infected, B cells are the natural interestingly, most of the cancer-associated proteins were reservoir of EBV (17). Moreover, immunomodulatory effects presented by class II molecules. In summary, this study are more likely to occur when the EBV infects B cells rather than suggests that EBV+ bystander B cells present in the tumor can epithelial cells since some tumor epithelial cells can interact with tumor-infiltrating T cells by acting as APC, thus downregulate the expression of HLA class I molecules (18). participating in the antitumor response and influencing the EBV can be maintained for life in hosts because it has the outcome of the patient. capacity to persist in a latent state. It is well known that EBV can take advantage of the immunosuppression status of individuals (19) as suggested by the increase of lymphoma incidence in HIV- infected and organ-transplanted patients (20). This was 2 MATERIAL AND METHODS supported by Mazar’s group who reported that 32% of patient- derived xenograft (PDX) tumors generated from epithelial tumor 2.1 Samples and Cell Cultures material can progress into EBV+ lymphocytic neoplasms (10)— 2.1.1 TNBC Biopsy or xenograft-associated B-cell lymphoma (XABCL) reported in The biopsy Breast Cancer-Patient Sample-562 (BC-PS-562) was other studies (21, 22)—evidencing the presence of EBV+ cells obtained from surplus hospital material, donated by the Vall within the tumor material. The NOD SCID gamma (NSG) mice, d’Hebron Institute of Oncology (VHIO), by standard procedures used for PDX generation, are likely more susceptible to EBV- with the appropriate approval of the Ethical and Scientific associated malignancies due to the lack of T-cell immune Committee of the institution. Consent was obtained from the responses. Our proposal is that the presence of EBV+ B cells patient according to local institutional review board requirement. within a tumor may shape the antitumoral immune response. The TNBC core biopsy was cut in several slices: five slices were Frontiers in Immunology | www.frontiersin.org 2 November 2021 | Volume 12 | Article 761798 Aran et al. EBV+ B Cells in Breast Cancer frozen in liquid nitrogen and seven slices were cultured on a 48- immunohistochemistry (IHC). Antibodies used for the paraffin- well plate. Biopsy-derived T-cell clones were cultured in 1 ml of embedded sections of the TNBC biopsy were anti-human CD3 Iscove’s modified Dulbecco’s medium (IMDM) GlutaMAX™ (clone 2GV6), anti-human CD4 (clone SP35), anti-human CD8 (Gibco) supplemented with 1% penicillin–streptomycin, 10% (clone SP57), and anti-human CD20 (clone L26) (Ventana of decomplemented human serum, and IL-2 (100 U/ml) Medical System, Inc.). IHC of the XABCL tissue was (provided by the NIH). For this study, only the three tissue performed in samples fixed in 10% neutral buffered formalin. frozen slices were used as a source of the original tumoral tissue, Transverse sections were used for processing. Morphological and the cultures derived from four of the slices were used to evaluation was performed on 5-µm paraffin-embedded sections, study the T cells in culture derived from the explants. stained with HE. The primary antibodies used were rabbit polyclonal anti-human CD3 (Dako) and rabbit polyclonal 2.1.2 XABCL Generation anti-human CD20 (Thermo Fisher). Sections were incubated PDX mice (PDX-562) were generated by implanting a TNBC with a labeled polymer [anti-rabbit for CD3 and CD20 (Agilent- (BC-PS-562) core biopsy into NOD SCID mice. After three Dako)] according to the instructions of the manufacturer. passes, the tumoral tissue was obtained. The tumor was IHC was completed using 3,3′-diaminobenzidine (DAB) and collected in PBS and disaggregated by using a scalpel. Crushed counterstaining in hematoxylin. Positive controls of the XABCL tissue was resuspended in 5 ml of DMEM/F12 (Gibco) IHC were performed using normal mouse spleen and lymph complemented with 10% of fetal bovine serum (Gibco), 1% L- node. In all the experiments, for the negative control, an isotype- glutamine (Biowest), 1% penicillin–streptomycin (Sigma- specific immunoglobulin was used to substitute the primary Aldrich), and 300 U/ml of collagenase type IA (Sigma-Aldrich) antibody as negative control; no immunostaining was detected and incubated twice in a shaker 80×g and 37°C during 30 min. in these sections. After centrifuging the disaggregated tissue, 100- and 40-mm pore cell strainers were used to obtain a uniform single-cell 2.3 Characterization of XABCL-LCL and suspension from the cell pellet. TNBC-T Cells 2.1.3 XABCL-LCL Culture 2.3.1 Phenotypic Characterization of T and B Cells After tissue digestion of the XABCL tumoral tissue, B cells were Between 2 and 5 × 105 of cells were stained with anti-human cultured and grown to a final concentration of 3 × 105 cells/ml in antibodies for 20 min at 4°C in the dark with PBS containing 2% T75 flasks using RPMI GlutaMAX™ (Gibco) with 1% penicillin FBS. After incubation, cells were washed twice, collected in and streptomycin (Sigma-Aldrich) and 10% of Fetal Bovine 200 ml of PBS, and analyzed by flow cytometry. Serum (Gibco). For the XABCL-LCL phenotype analysis, anti-human antibodies used were PE-conjugated anti-CD19 (BD 2.1.4 TNBC-T Cells and XABCL-LCL Co-Cultures Pharmingen), PE-conjugated anti-CD20, FITC-conjugated To select XABCL-reactive T cells, TNBC-T cells from one of the anti-CD21, PE-Cy7-conjugated anti-CD38, APC-conjugated tissue slices were grown for 10 days in culture medium without anti-HLA-ABC (BD Pharmingen), PE-conjugated anti-HLA- cell-specific stimulus. Cells were grown at a final concentration of DR (BD Pharmingen), FITC-conjugated anti-CD80 3 × 10 5 cells/ml with IMDM GlutaMAX ™ (Gibco) (ImmunoTools), FITC-conjugated anti-CD86 (ImmunoTools), complemented with 10% of human serum, 1% penicillin– PE-conjugated anti-PD-1 (eBioscience), and FITC-conjugated streptomycin (Sigma-Aldrich), and IL-2 at 100 U/ml in contact anti-PD-L1 (BD Pharmingen). For the T-cell phenotype analysis, with XABCL-LCL B cells and allogeneic PBMCs, previously the following human antibodies were used: PerCP-conjugated irradiated at 60 and 30 Gy, respectively. On the 7–10th day, anti-human CD3, PE-conjugated anti-human or FITC- cells were restimulated using anti-human CD3 and anti-CD28- conjugated anti-human CD4, and FITC-conjugated anti- coated Dynabeads (Gibco). As a control, cells were grown using a human CD8 or APC-conjugated anti-human CD8 (BD rapid expansion method (REM) (26). T cells (1–1.5 × 105) were Pharmingen). The flow cytometer used was BD FACS Canto. cultured in the presence of feeders (25 × 106 30 Gy irradiated Analyses were performed by using the FlowJo and FACS PBMCs) in a T25 flask with 25 ml of complete medium (IMDM Diva software. GlutaMAX™ with 1% P/S and 10% of decomplemented human serum) supplemented with 50 ng/ml of soluble anti-CD3 2.3.2 BCR and TCR High-Throughput Sequencing of (OKT3) and IL-2 at 200 U/ml. On the fifth day, half of the TNBC, XABCL, TNBC-T Cells, and XABCL-LCL medium was removed and replaced with fresh medium with IL-2 DNA extraction from the TNBC and XABCL tissues and at 200 U/ml, and cells were collected 10–12 days XABCL-LCL was carried out using a phenol–chloroform after stimulation. protocol. Tissue samples were disrupted by sonication. Amplification of BCR and TCR transcripts from DNA was 2.2 Tumoral and T- and B-Cell Presence in performed using survey level ImmunoSEQ technology by the Biopsy Adaptive Biotechnologies. For the high-throughput sequencing 2.2.1 Immunohistochemistry (HTS) of TNBC-T cells, cells were counted and between 1 × 105 The presence of tumoral cells and infiltrating T and B cells in the and 1 × 106 of T cells from the initial cultures and expanded T biopsies was determined by hematoxylin and eosin (HE) and cells were used for RNA extraction. RNA was isolated using Frontiers in Immunology | www.frontiersin.org 3 November 2021 | Volume 12 | Article 761798 Aran et al. EBV+ B Cells in Breast Cancer RNeasy Micro Kit and RNeasy Mini Kit (Qiagen), respectively, HCl (pH 8.0), 150 mM NaCl, 1× Complete protease inhibitor following the instructions of the manufacturer. An optional on- (Roche), and 5 mM EDTA]. Solubilized pelleted membranes column DNase digestion was also performed during the RNA were sonicated 4× 5 s (20% amplitude of max. 130 W) and isolation using the RNase-Free DNase Set (Qiagen). RNA incubated overnight at 4°C. Non-solubilized membranes were amounts and integrity were measured with Agilent 2100 pelleted by ultracentrifugation at 55,000×g for 1 h at 4°C, and bioanalyzer (Agilent Technologies). Human TCR profiling HLA complexes in the supernatant were then purified from the from TIL was assessed by using the SMARTer Human TCR a/ soluble fraction based on the Spetniak et al. protocol (35). b Profiling Kit (Takara Bio), which allows the complete capture Solubilized membranes were incubated overnight with 100 ml of V(D)J variable regions of TCR transcripts and library CNBr-activated sepharose beads (GE Healthcare Life Sciences) production, directly indexed for sequencing. Both TCR-alpha coupled to anti-HLA-ABC w6/32 or to the anti-HLA-DR B8.11.2 and TCR-beta chain diversities were studied. Purification of antibodies. Antibody-conjugated sepharose beads were washed amplified libraries was performed using Agencourt AMPure 3× with 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, and 0.5% n- XP Beads (Beckman Coulter), following the commercial dodecyl-b-D-maltoside and 3× with 50 mM Tris–HCl (pH 8.0) recommendations. Analysis and validations of libraries were and 150 mM NaCl. Unspecific interactions were diminished by carried out on an Agilent 2100 Bioanalyzer, using the DNA washing with a high-salt concentration buffer [50 mM Tris–HCl 1000 Kit (Agilent Technologies). Sequence was performed on an (pH 8.0), 0.5 M NaCl]. Peptide–HLA–DR complexes were eluted Illumina MiSeq sequencer using the 600-cycle MiSeq Reagent Kit with 0.25% TFA after washing 3× with 50 mM Tris–HCl (pH v3 (Illumina) with paired-end 2 × 300 base pair reads. 8.0) and 150 mM NaCl and 1× with 20 mM Tris–HCl (pH 8.0). The eluted complexes were cleared by SCX and a C18 tip before 2.3.3 TCR and BCR Repertoire Analysis mass spectrometry. TCR and BCR sequencing results obtained from the ImmunoSEQ analysis were evaluated by the ImmunoSEQ 2.4.2 Peptide Identification by Mass Spectrometry analyzer, v.3.0. TCR sequencing data obtained from the Samples were analyzed by liquid chromatography coupled to SMARTer Human TCR a/b Profiling Kit were evaluated by the high-resolution mass spectrometry (LC-HRMS). The LTQ MiXCR Immune Repertoire Analyzer (27). Raw data obtained Orbitrap XL mass spectrometer (Thermo Fisher) equipped from both technologies were processed using VDJTools (28). with a nanoESI source and coupled to an Agilent 1100 Non-productive clonotypes were excluded. The remaining nanochromatographic system was used. LC separation was repertoire data were collapsed by CDR3 amino acid sequences, performed using a 140-min acetonitrile gradient. Analyses i.e., clonotypes with different nucleotide sequences encoding the were performed in data-dependent mode at a target mass same CDR3 amino acid sequence were summed, and frequencies resolution of 60,000 (at m/z 400). Up to 10 of the most intense were recalculated. The different TCR and BCR sequencing results peaks with charge ≥2 and intensity above the 500-unit threshold from each sample were compared to find out the common were selected and fragmented by CID. To minimize the sequences shared by the XABCL, TNBC, and TNBC-T cells. redundant selection of precursor ions, dynamic exclusion was Diversity and heat plot analyses were also performed using set to 1 for 45 s. Raw data were processed using Proteome VDJTools (28). The TCR sequences obtained from this Discoverer 1.4 against the human and viral proteome database analysis were compared with the McPAS-TCR (29) database (UniProt/SwissProt) with the following parameters: no enzyme, with a Levenshtein distance of 1. Figures were performed using 20 ppm precursor mass tolerance, 0.02 Da fragment tolerance, the Graphpad Prism 7.0 software. UpSet plots were generated and variable modification of oxidized methionine (+16 Da). using R (version 3.6.1) (30) using the “dplyr,” (31) Peptide spectral matches were filtered at 1% FDR “RColorBrewer,” (32) and “UpSetR” (33) packages. using Percolator. 2.4 Characterization of the HLA- Associated Peptide Repertoire 2.4.3 HLA-Derived Peptide Repertoire Analysis From the XABCL-LCL Peptides identified were prefiltered by following these rules: 2.4.1 Purification of Peptide–HLA Complexes elimination of I) peptides outside the range 8–13mer and Two 40 × 106 pellets of XABCL-LCL were used to elute peptides peptides shorter than 10-mer peptides, for class I and class II presented by HLA-ABC and HLA-DR. The cellular lysis was analyses, respectively; II) keratin-derived peptides; and III) performed as described by Heyder et al. (34) Briefly, the cell duplicate peptides with modifications. The subcellular location pellet was resuspended in lysis buffer [50 mM Tris–HCl (pH 8.0), of the source proteins was extracted from the UniProt (36) 150 mM NaCl, 1× Complete protease inhibitor (Roche), and database. Proteins that were found in more than one 5 mM EDTA]. Nuclei and cell debris were cleared by 2× 12 min subcellular location were counted in both localizations. centrifugations at 5,500×g after sonication (40% of amplitude of DAVID (37, 38), which executes a functional annotation max. 130 W) and 10 min incubation at 4°C. Membranes clustering using different databases, was used for the KEGG collected from the supernatant were pelleted with 1 h Pathway Database pathway enrichment analysis. For the Gibbs ultracentrifugation at 72,000×g and 4°C and resuspended in a clustering, peptides found in nested sets were excluded, leaving solubilization buffer [1% n-dodecyl-b-D-maltoside, 50 mM Tris– only the shortest peptide. Frontiers in Immunology | www.frontiersin.org 4 November 2021 | Volume 12 | Article 761798 Aran et al. EBV+ B Cells in Breast Cancer 2.4.4 Identification of Proteins in the XABCL-LCL cells associated with Hodgkin’s lymphoma (12). An HLA HLA Peptide Repertoire genotyping confirmed the patient cells as the origin of the To exclude peptides from proteins commonly presented by LCL, XABCL tumor (HLA-A *02:01, A*30:02, B*18:01, B*49:01, the source proteins of the peptides obtained in the HLA-ABC Cw*05:01, Cw*12:03, DRB1*03:01, DRB1*11:01, DQB1*02:01, analysis were compared with 26,305 source proteins, from 6 DQB1*03:01), and we confirmed that both the XABCL-LCL and different studies and 7, 11, and 9 different HLA-A, B, and C the PBMCs from the patient—but not the TNBC-T cells—were alleles, respectively (39–44). For the HLA-DR peptides, 517 EBV+ by qPCR (data not shown). The phenotypic analysis of the source proteins from 10 different studies and 11 different XABCL-LCL revealed a molecular pattern that resembled an HLA-DR alleles found in the literature (45–54) were used. The activated germinal center B cell, i.e., CD19+, CD20+, CD21−, assignment of the peptides to the corresponding allele was and CD38+, and they also expressed HLA-ABC and HLA-DR performed according to the following criterion. First, only molecules as well as CD80, CD86, and PD-L1 (Supplementary peptides clustered by Gibbs Cluster 2.0 (55) were used. Then, Figure 1), confirming that these cells have antigen-presenting peptides predicted as strong binders by the NetMHCIPan 4.1 capacities, as it was expected in a B-cell line. We could not detect Server and the NetMHCIIpan 4.0 Server (56) were considered. antibodies in the supernatant from the XABCL-LCL culture Peptides with a peptide spectral match (PSM) of 1 were excluded. (data not shown). The source proteins from the peptides obtained were examined A BCR HTS revealed that one single BCR sequence, i.e., in the Human Protein Atlas (57–59) to check for specificities in CAKDVTDVSAYYYQAEYFPHW (IGHV03-23), represented cancer. The number of proteins, peptides, and PSM was more than the 97% of the repertoire in both samples, i.e., the considered. All data were represented using the GraphPad XABCL tissue and the XABCL-LCL (Supplementary Table 1), Prism 7.0 software. confirming a monoclonal expansion produced before the in vitro cell line establishment. We performed the BCR HTS on the TNBC tissue and the same BCR sequence was found, confirming that the B cells that composed the XABCL were originally 3 RESULTS present within the tumor infiltrate of the patient and transformed into a monoclonal lymphocytic tumor in the mouse. 3.1 The Tumor Generated in a PDX Derived From the Tumor of a TNBC 3.2 XABCL B Cells Can Maintain Tumor- Patient Was the Result of a Monoclonal Infiltrating T Cells In Vivo and In Vitro Expansion of EBV+ Tumor-Infiltrating Tissue sections were obtained from each pass in mice and the B Cells IHC of the XABCL tissue revealed some CD3+ T cells A 44-year-old patient was diagnosed with TNBC. The presence infiltrating the tumor after the first pass (Supplementary of an infiltrating ductal carcinoma [histological grade 3, Figure 2). A TCR CDR3 HTS was performed to study which no vascular invasion, estrogen receptor (ER)-negative, infiltrating T-cell clonotypes could have remained in the progesterone receptor (PR)-negative, human epidermal growth XABCL tissue, obtained from mice after the third pass. factor 2 (HER2)-negative, and Ki67 70%] was confirmed by the The analysis revealed 43 different T-cell clonotypes where the Pathological Anatomy Unit of the Vall d’Hebron Institute of most prevalent clone represented 31.8% of the sample Oncology (Barcelona, Spain). (Supplementary Table 2). Two core biopsies were obtained at the same time, i.e., To prove that the clonotypes found in the XABCL tissue came 2 weeks after diagnosis and before neoadjuvant chemotherapy; from the human biopsy, a TCR CDR3 HTS was also realized on one was inoculated in an NSG mouse to obtain a PDX and the the TNBC samples. As expected, many more clonotypes were other one was used to study the T-cell infiltration directly from identified: 26,724 from the TNBC and 5,451 in the TNBC-T cells. the tissue or from the T-cell cultures (TNBC samples, from now A total of 1,144 clonotypes were shared between these two sets on TNBC and TNBC-T cells, respectively). The TNBC (Figure 3A and Supplementary Table 2). Eighteen TCR (Figure 1A) was infiltrated with CD20+ B cells and CD3+ T sequences appeared in common between the TNBC samples cells (Figures 1B, C). A similar distribution was observed for and the XABCL tissue: 4 were shared by three samples, 13 were both B and CD4+ T cells, suggesting that these cells could be shared by TNBC and XABCL tissues, and only 1 sequence was interacting in the tumor site. Although there was a higher exclusively found in the TNBC-T cells and the XABCL tissue. presence of CD4+ T cells (Figure 1D), CD8+ T cells were also Only one of the four TNBC-T cell samples (slices in culture) found (Figure 1E). analyzed (see Material and Methods) shared a TCR sequence The tumor grown in the PDX mouse was obtained after three with the XABCL. passes in mice. Cells from this culture did not show epithelial A tracking heat plot of the 50 most abundant clonotypes— tumor cell line characteristics, but an LCL-like phenotype. A using samples with shared sequences—revealed a different stable long-term cell line was established from the digested pattern of TCR in the three sets, especially in the XABCL tumoral tissue (XABCL-LCL, from now on). The lymphocytic tissue (Figure 3B), i.e., the most abundant clonotypes origin was confirmed by IHC (Figure 2), and not only most infiltrating the XABCL after 6 months were not the most of the cells were CD20+, but also some of these cells presented abundant in the TNBC tissue or in the TNBC-T cell sample. a bilobed nucleus, reminding of the Reed–Sternberg Thus, the XABCL model demonstrated that B cells can maintain Frontiers in Immunology | www.frontiersin.org 5 November 2021 | Volume 12 | Article 761798 Aran et al. EBV+ B Cells in Breast Cancer A B C D E 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). some tumor-infiltrating T cells, confirming a possible role of B TNBC-T cells expanded in the presence of OKT3 (anti-human cells as APC in the tumor microenvironment. CD3). The TCR repertoire and T-cell phenotypic changes after In vitro stimulation was performed by co-culturing TNBC-T co-culture were analyzed by TCR HTS and flow cytometry, cell with irradiated XABCL-LCL and comparing with control respectively. A low number of sequences (73 clonotypes) with very low TCR diversity were obtained after co-culture (Figure 4A and Supplementary Table 3), also displaying a pattern different from the OKT3 control (Figure 4B and Supplementary Figure 3A). Only two of the five shared sequences between XABCL tissue and TNBC-T cells (Figure 3A) were maintained, i.e., CARSTNGELFF (TRBV28) and CSARVPGLPDTQYF (TRBV20-1) (Supplementary Figure 3B). These two clonotypes were also found in the control T-cell cultures expanded with anti-CD3, but the CARSTNGELFF sequence represented a larger normalized fraction after the co-culture with the XABCL-LCL. Phenotypic analysis (Supplementary Figure 3C) showed little differences other than a decrease of the double-negative (DN) T-cell population, which represented 41.8% of T cells in the initial culture and only 1.34% after co- culture. This subset appeared to be replaced by CD4+ T cells, varying from 15% in the initial culture to 43.6% after co-culture. CD8+ T cells displayed similar frequencies in both samples: FIGURE 2 | The tumor derived from a TNBC patient implanted in a PDX was 40.6% and 52.1% in the initial culture and after co-culture, a B-cell lymphocytic tumor (XABCL). CD20 immunohistochemical stain in the respectively. Thus, the results of the in vitro experiments histological section revealed that most of the cells were CD20+ B cells. Cells confirmed that, even after transformed to an LCL, B cells with a bilobed nucleus, reminding of the Reed–Sternberg cells associated derived from the XABCL tumor influenced the TCR repertoire with Hodgkin’s lymphoma, were observed (pointed with a red arrow). of the infiltrating T cells. Frontiers in Immunology | www.frontiersin.org 6 November 2021 | Volume 12 | Article 761798 Aran et al. EBV+ B Cells in Breast Cancer A B 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. 3.3 Previously Reported TCR Sequences sequences (CASSGGGAYNEQFF and CASSIQETQYF) were found Are Maintained by XABCL B Cells in the three sets, but it is particularly interesting that the Clonotypes selected both in vivo (43 from XABCL tissue) and in vitro CASSIQETQYF sequence had been reported 6 times in (73 after XABCL-LCL co-culture) were investigated in the TCR autoimmunity, 3 times in infections, and 17 times in cancer, and database Mc-PAS-TCR (29) (Figure 5 and Supplementary in this latter group, it was described as a neoantigen- Table 4). None of the five common clonotypes between XABCL reactive clonotype. tissue and TNBC-T cells had been previously reported in the literature. Ten of the 43 clonotypes infiltrating the XABCL tissue 3.4 HLA-Associated Peptides Presented had been reported; all sequences were described as pathogen- by the XABCL-LCL reactive TCR, although some were also related to autoimmunity The HLA peptidome from XABCL-LCL was analyzed by mass (two sequences) and cancer (two sequences). One of these sequences spectrometry. We identified 1,572 HLA-ABC-associated peptides was identified in the three categories (CASSLEGDQPQHF). and 1,004 HLA-DR-associated peptides, derived from 1,090 and Thirteen of the 73 in vitro expanded clonotypes were found in the 444 source proteins, respectively (Supplementary Table 5). No database: 10 were reported as pathogen-reactive, 2 sequences were EBV-derived peptide was found. After excluding duplicated exclusively found in autoimmunity, and 1 was found in cancer. Two peptides, i.e., equal sequences with modifications, selecting by Frontiers in Immunology | www.frontiersin.org 7 November 2021 | Volume 12 | Article 761798 Aran et al. EBV+ B Cells in Breast Cancer the HLA-DR peptidome. A clustering analysis of the peptides was performed by Gibbs (55), revealing different motifs matching the A HLA alleles of the XABCL-LCL sample. We analyzed the XABCL-LCL immunopeptidome, and to exclude commonly presented proteins, the peptide repertoire was compared with LCL peptide repertoires described in the literature from different HLA types. Six studies (39–44) were selected from the literature describing the repertoire of HLA- ABC-derived peptides in LCL (from 7, 11, and 9 different HLA- A, B, and C alleles, respectively). The total number of source proteins used was 26,305. Peptides from the XABCL-LCL were filtered, excluding those derived from proteins that were previously identified in the LCL database generated. The same procedure was performed with the HLA-DR-derived peptidome, and the LCL database generated contained 517 source proteins and 11 different HLA-DR alleles from 10 different studies (45– 54). We then analyzed the predicted binding affinities of the B filtered peptides (see Material and Methods) and selected only those predicted as strong binders. We finally selected 195 peptides predicted as strong binders to HLA-ABC alleles and 223 to HLA-DR alleles. These filtered sequences corresponded to 178 and 102 different proteins, representing 16% and 23% of the total source proteins from the class I- and class II-derived peptides, respectively, obtained from the XABCL-LCL. The KEGG pathway analysis of the source proteins [DAVID (37, 38)] showed that the top 2 results were the antigen processing and presenting (p = 1.14E-4) and the phagosome pathways (p = 6.6E-3), indicating that the major role of these B cells was antigen presentation. Finally, the association with cancer of the remaining proteins was checked in the Human Protein Atlas (57–59). For this purpose, the identified proteins and peptides and the number of spectra identified for each peptide (PSM) were used. A higher PSM value is related to a higher abundance of the presented peptide. Most of the analyzed proteins could be found or FIGURE 4 | TNBC-T cells expanded in vitro in the presence of XABCL-LCL enriched in many cancer tissues, named as low cancer-specific presented a highly restricted TCR repertoire with a different pattern. (A) The proteins (Figure 6 and Supplementary Table 6). In the HLA- TCR repertoire size and diversity hugely decreased in the TNBC-T cell sample ABC repertoire, there were only two cancer-enriched proteins expanded in the presence of irradiated XABCL-LCL (60 Gy), compared with and a high number of proteins without enough available data in 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 the database. Interestingly, several proteins specific for some expanded TBNC-T cells was also different in the T cells grown with XABCL- cancers such as thyroid cancer (1 protein, 8 peptides, and 280 LCL, instead of with OKT3, as several sequences were not found in common PSM), glioma (2 proteins, 6 peptides, and 203 PSM), and liver and different frequencies of the shared clonotypes were observed cancer (3 proteins, 6 peptides, and 146 PSM) were identified in (Supplementary Table 3). the HLA-DR peptide repertoire. length and excluding keratin-derived peptides, 1,392 (HLA-ABC) 4 DISCUSSION and 834 peptides (HLA-DR) derived from 1,072 and 353 source proteins remained. Selected peptides fit well to the class I and class II Tumors impair the immune system action generating an adverse peptide length patterns (Supplementary Figure 4). Similarly, the microenvironment by expressing immune checkpoints that overall subcellular location of the source proteins given by UniProt (36) favor a regulatory immune response (60). ICPI treatments such as corresponded to the different HLA pathways, revealing more source pembrolizumab or nivolumab and ipilimumab against PD-1 and proteins derived from the membrane and the endocytic pathway in CTLA-4 receptors, respectively, have increased the survival of the HLA-DR- compared to the HLA-ABC-associated peptides cancer patients (61). At the same time, blocking the immune (Supplementary Figure 4). Peptides forming nested sets, i.e., checkpoint molecules can trigger the disruption of immune peptides sharing a common sequence with varying flanking homeostasis and, consequently, lead to immune-related adverse residues, a classical feature of class II peptides, were observed in events [known as irAEs (62)]. The infection by some human Frontiers in Immunology | www.frontiersin.org 8 November 2021 | Volume 12 | Article 761798 Aran et al. EBV+ B Cells in Breast Cancer 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. viruses itself has also been proposed to be related with the tumor landscape. The role of B cells as a putative inductor of autoimmune and inflammatory processes in several cases (24, the T-cell response in the tumoral microenvironment in BC is 25, 63). Thus, the presence of EBV+ B cells, as well as other virus- still unclear (67). infected cells, in an immunomodulated microenvironment The XABCL generated from the TNBC biopsy resulted in a together with ICPI treatment may be an additional factor with a lymphocytic tumor; XABCL-LCL was CD19+, CD20+, CD21−, role in the development of irAEs. and CD38+, an activated germinal center B-cell phenotype. We In the last years, different groups have performed analysis of confirmed that the XABCL-LCL, as well as the PBMCs of the the association between infection by different species of human patient, was EBV+. This is not surprising since up to 40% of herpesvirus (HHV) such as EBV (or HHV-4) (64) and Hodgkin’s lymphomas are EBV-associated and LCLs can be cytomegalovirus (CMV or HHV-5) (65), or retrovirus such as generated in vitro using EBV. Considering that EBV is detected in human T-lymphotropic virus (HTLV) (66), and the tumor more than 90% of the adult population of the world (68) and that it outcomes. However, these studies are all focused on the direct has the ability to be maintained for life in a latent stage, it is not role of the viruses in the development of tumors but not in the unexpected that among TIL there were EBV+ B cells. The contribution of the antivirus responses to the antitumor development of EBV+ lymphocytic neoplasms in PDX derived response. In this research, we have been able to study different from breast cancer had also been previously described (10). This can immune-related factors associated with the presence of EBV+ B be explained by the lack of a mature immune system in the PDX cells among TIL. We have studied a TNBC biopsy obtained models, which are NSG mice, and the evidence that EBV infection before neoadjuvant chemotherapy and a XABCL lymphocytic more frequently leads to lymphoproliferative disorders in tumor generated from a simultaneously obtained biopsy, as well immunosuppressive contexts (20, 69, 70). We are not aware that as T cells derived from the TNBC (TNBC-T cells) and the EBV+ these XABCLs have been used before as a model to investigate the B cell line derived from the XABCL (XABCL-LCL). role of tumor-infiltrating EBV+ B cells. IHC of the biopsy of the patient first revealed that most of the The BCR analysis of the XABCL revealed a unique BCR CD3+ T cells found in the histological section were CD4+ T cells sequence representing the major fraction in both the tissue and colocalized with CD20+ cells, while CD8+ T cells were localized the LCL. This confirmed that the conversion into a lymphocytic nearest to the tumor in the infiltrating front of neoplasia. This tumor was produced in the mice and not by the establishment of would suggest a possible in situ interaction between B cells and an LCL in vitro. We analyzed the BCR repertoire in the initial Th. Since B cells can act as professional APC, the presence of B patient biopsy, and this BCR sequence was found in the TNBC, cells, even if they are not tumor-specific, can contribute to tune among many other BCR sequences. Frontiers in Immunology | www.frontiersin.org 9 November 2021 | Volume 12 | Article 761798 Aran et al. EBV+ B Cells in Breast Cancer increased expression of the HLA molecules is usually observed in EBV+ B cells (63). Moreover, a different pattern of TCR was observed in the TNBC samples compared with the XABCL, suggesting that certain clonotypes were being selected by XABCL B cells. Therefore, we used TNBC-T cells for co-culture with irradiated XABCL-LCL to study if a T-cell selection could also be observed in vitro. Upon interaction with the XABCL-LCL, 73 clonotypes were identified and a decrease in TCR diversity was observed. Two of the sequences expanded were previously found in the XABCL, i.e., CARSTNGELFF (TRBV28) and CSARVPGLPDTQYF (TRBV20-1). Certain clonotypes found in the initial cultures were maintained in the XABCL-LBL- expanded TNBC-T cells, but not in the control. Thus, these results altogether suggest that some EBV+ B cells may be able to stimulate T cells from TIL, inducing a certain selection of the TCR repertoire. The peptide repertoire presented by HLA-ABC and HLA-DR in the XABCL-LCL was analyzed. We did not find EBV-derived peptides neither in the class I nor in the class II analysis. This is not very surprising since EBNA1 is the only viral protein expressed in all EBV-associated malignant diseases (71). The subcellular location of source proteins showed a pattern common to B-cell line repertoires (53, 54). The clustering analysis obtained revealed two binding motifs matching the HLA haplotype of the patient. It is worth mentioning that in the HLA-DR study, the most abundant source protein was GAPDH, and although it is a commonly found protein, it is not that frequent to find HLA-DR-associated GAPDH peptides in high abundance. A relationship between GAPDH with breast cancer has been described (72, 73); therefore, it could be interesting to further analyze the relevance of the GAPDH- derived peptides presented by these cells. There are several studies of the HLA class I and class II repertoire using LCL, but these are transformed in vitro while the FIGURE 6 | Distribution of the selected proteins obtained from the XABCLLCL HLA repertoire based on their expression in cancer. After XABCL B cells used in this study were infected and transformed comparing the source proteins with those found in the literature, the by the EBV in vivo. Therefore, there could be differences in the expression in cancer of 178 (HLA-ABC-associated) and 102 (HLA-DR- peptide repertoire between these cells and the typical LCL. In associated) proteins was checked by the Human Protein Atlas. Counts of order to study this, we excluded the source proteins from the proteins, peptides, and PSM obtained are represented. The highest count in peptides obtained from LCL found in the literature, all the measures corresponded to proteins with low cancer specificity, i.e., proteins enriched in many cancer tissues. Two proteins from HLA-ABC- independently for the class I (39–44) and the class II alleles derived peptides showed specificity in certain cancers. Regarding the HLA- (45–54). The resulting protein pool represented 16% and 23% of DR results, peptides with high PSM values corresponded to specific proteins the total source proteins obtained from the HLA-ABC and the from thyroid cancer (280 PSM and 8 peptides contained in 1 nested set and HLA-DR peptidomes, respectively. These proteins were mostly 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). involved in the antigen processing and presentation and in the phagosome pathways. This indicates that these cells may present some different proteins compared with other LCL and that these Interestingly, the histological section obtained from the first B cells still can act as APC. pass of the XABCL tumor revealed some CD3+ T cells Many of the selected sequences were derived from proteins infiltrating the tumor. TCR sequencing confirmed that some T that can be present in many cancer tissues, catalogued as proteins cells were still maintained after the third XABCL pass. Forty- with low cancer specificity. Nevertheless, some proteins enriched three clonotypes were obtained and 18 of these were also found in cancer were also found, especially in the HLA-DR selected in the TNBC frozen tissue or in the expanded TNBC-T cells. The peptides. Thus, in a tumoral microenvironment, peptides derived maintenance of T cells in the XABCL during the three passes in from autologous proteins as cancer-enriched proteins can be mice may be the result of stimulation by the XABCL B cells, presented by these B cells. In the future, these peptides should be which expressed the HLA class I and class II molecules as well as used separately in vitro to analyze their capacity to stimulate the costimulatory molecules CD80, CD86, and PD-L1. An cancer-infiltrating T cells. Frontiers in Immunology | www.frontiersin.org 10 November 2021 | Volume 12 | Article 761798 Aran et al. EBV+ B Cells in Breast Cancer Several features of EBV+ B cells make them relevant in the ProteomeXchange Consortium via the PRIDE (77) partner tumor context. Because tumor cells are known to downregulate repository with the dataset identifier PXD028646. the HLA molecules to escape from immune response, the activation and maintenance of both CD4+ and CD8+ T cells in the tumor site is usually the responsibility of APC. The role of B cells as modulators of the antitumoral response has been latterly AUTHOR CONTRIBUTIONS considered (74). However, EBV+ B cells in a latent stage are Conceptualization: AA and MM. Methodology: AA, MM, CR-M, memory-like B cells activated by signaling pathways similar with MC, EM, and YA. Investigation: AA, VP, RR, EM, and YA. those produced by the CD40L-mediated activation (75), in the Resources: VP, RR, CB, EZ, JA, and MC. Writing—original draft: absence of T-cell signals and regardless of their BCR specificity. AA and MM. Writing—review and editing: AA, MC, JC, and Moreover, EBV+ B cells in a latent stage have an abnormal MM. Supervision: JC and MM. All authors contributed to the transcriptome (76). On the other hand, although EBV-specific T article and approved the submitted version. cells are maintained throughout life, participating in a persistent control of the infection, the immunomodulatory capacities of the tumor cells affecting the microenvironment may give the EBV a FUNDING chance to start an infectious stage and to escape the immune response. Two recent studies have reported fatal inflammations This project was funded by Roche Farma, S.A. grant [encephalitis (25) and myocarditis (24)] in post-ICPI melanoma SP181123001 and the Spanish Ministry of Science, Innovation patients, in which EBV-specific T cells were involved. Even so, and Universities grant RTI2018-097414-B-I00. Partial financial the antigens responsible for these dysregulations may not be support was received from the “El Paseı́co de la Mama” 2015. limited to EBV-derived antigens. This study received partial funding from Roche Farma, S.A. The In summary, this is a first proof of concept that gives funders were not involved in the study design, collection, relevance to EBV+ B cells present in the tumor site. We have analysis, interpretation of data, the writing of this article, or not only demonstrated their presence but also that they are in an the decision to submit it for publication. activated status and able to stimulate T cells from TIL. Our data also evidence that EBV+ B cells can modulate the TCR repertoire as a certain pattern has been identified and some of the TCR ACKNOWLEDGMENTS sequences maintained had been already reported in the literature. Certain uncommon peptides presented by HLA- We want to particularly thank the patient for participating in this ABC and HLA-DR molecules have been also identified. Thus, study. We thank the SCAC and the SGB services of the IBB-MRB to take profit of the XABCL tumors can be a tool to understand (UAB, Bellaterra), especially Manuela Costa, for the help in the the role of EBV+ B cells in the tumor infiltrates and how they can experimental procedures. We acknowledge A. Gonzá lez, Ph.D., contribute to different patient outcomes by shaping the and M. Juan, M.D., Ph.D., from Hospital Clinic Barcelona TCR repertoire. (Barcelona) for the support in the BCR sequencing. We also want to acknowledge Prof. D. Jaraquemada from the Immunology Unit (Cellular Biology, Physiology and Immunology Department, UAB, Bellaterra) for the help in the DATA AVAILABILITY STATEMENT peptide analysis and for the thorough revision of the manuscript. The datasets presented in this study can be found in online repositories. TCR sequencing data from the TNBC-derived T cell cultures are available as SRA accessions in the BioProject SUPPLEMENTARY MATERIAL PRJNA75917. The TCR and BCR HTS from TNBC and XABCL biopsies are available as an ImmuneAccess Project The Supplementary Material for this article can be found online (DOI https://doi.org/10.21417/AA2021FI). The mass at: https://www.frontiersin.org/articles/10.3389/fimmu.2021. spectrometry proteomics data have been deposited to the 761798/full#supplementary-material REFERENCES 3. Kuroda H, Jamiyan T, Yamaguchi R, Kakumoto A, Abe A, Harada O, et al. Tumor-Infiltrating B Cells and T Cells Correlate With Postoperative 1. Gao G, Wang Z, Qu X, Zhang Z. Prognostic Value of Tumor-Infiltrating Prognosis in Triple-Negative Carcinoma of the Breast. 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