Tumor-Infiltrating B Lymphocyte Profiling Identifies IgG-Biased, Clonally Expanded Prognostic Phenotypes in Triple-Negative Breast Cancer

Abstract

In breast cancer, humoral immune responses may contribute to clinical outcomes, especially in more immunogenic subtypes. Here, we investigated B lymphocyte subsets, immunoglobulin expression, and clonal features in breast tumors, focusing on aggressive triple-negative breast cancers (TNBC). In samples from patients with TNBC and healthy volunteers, circulating and tumor-infiltrating B lymphocytes (TIL-B) were evaluated. CD20⁺CD27⁺IgD^- isotype-switched B lymphocytes were increased in tumors, compared with matched blood. TIL-B frequently formed stromal clusters with T lymphocytes and engaged in bidirectional functional cross-talk, consistent with gene signatures associated with lymphoid assembly, costimulation, cytokine-cytokine receptor interactions, cytotoxic T-cell activation, and T-cell-dependent B-cell activation. TIL-B-upregulated B-cell receptor (BCR) pathway molecules FOS and JUN, germinal center chemokine regulator RGS1, activation marker CD69, and TNFα signal transduction via NFκB, suggesting BCR-immune complex formation. Expression of genes associated with B lymphocyte recruitment and lymphoid assembly, including CXCL13, CXCR4, and DC-LAMP, was elevated in TNBC compared with other subtypes and normal breast. TIL-B-rich tumors showed expansion of IgG but not IgA isotypes, and IgG isotype switching positively associated with survival outcomes in TNBC. Clonal expansion was biased toward IgG, showing expansive clonal families with specific variable region gene combinations and narrow repertoires. Stronger positive selection pressure was present in the complementarity determining regions of IgG compared with their clonally related IgA in tumor samples. Overall, class-switched B lymphocyte lineage traits were conspicuous in TNBC, associated with improved clinical outcomes, and conferred IgG-biased, clonally expanded, and likely antigen-driven humoral responses. SIGNIFICANCE: Tumor-infiltrating B lymphocytes assemble in clusters, undergoing B-cell receptor-driven activation, proliferation, and isotype switching. Clonally expanded, IgG isotype-biased humoral immunity associates with favorable prognosis primarily in triple-negative breast cancers.

Graphical abstract

Figure 1.

Flow cytometric analyses reveal reduced circulating CD20⁺CD27⁺ memory and amplification of tumor-infiltrating CD20⁺CD27⁺IgD⁻ class-switched subsets among B lymphocytes. A, Gating strategy for identification of B lymphocytes and memory (B^m) lymphocytes derived from peripheral blood mononuclear cell (example patient peripheral blood mononuclear cell shown). B, Quantification of total circulating B cells (top) and B^m cells (bottom) as percentage of CD45⁺ cells in HV (N = 48) and patient (N = 55) peripheral blood (KCL flow cohort; Supplementary Table S1 for patient information), stratified according to stage and treatment status. C, Quantification of B lymphocytes (CD20⁺) from single cell suspensions of normal breast (N = 9), NANT (N = 12), and cancer tissue (N = 17) samples. D, Quantification of matched patient circulating- and tumor-infiltrating CD20⁺CD27⁺ B cells (matched samples of 7 patients). E, Quantification of CD20⁺CD27⁺IgD⁻ B cells in HV (N = 17), patient peripheral blood (N = 7), and cancers (N = 8) of total CD20⁺CD27⁺ B cells. Statistical significance was determined using the Student t test. ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 2.

B lymphocyte infiltration and its positive prognostic value in TNBC. A, TIL-B quantification by IHC within normal breasts and TNBC (N = 15 each, Bart's IHC cohort), and by GEx (normal breast) vs. TNBC (N = 10 vs. 131, KCL GEx cohort); non-TNBC vs. TNBC (N = 515 vs. 123, TCGA GEx cohort); TNBC subtypes [mesenchymal (M), luminal androgen receptor (LAR), basal-like 1 and 2 (BL1 and 2), and immunomodulatory (IM; N = 122, KCL GEx cohort]. The Mann–Whitney test was used for statistical significance. B, TNBC TIL-B correlation with TIL-T by IHC (r = 0.72, Bart's IHC cohort) and by GEx (r = 0.73, KCL GEx cohort). Linear regression analysis was used to calculate correlation coefficients (r) and P values. C, B lymphocyte metagene NanoString GEx data comparing B lymphocytes in primary tumors with metastatic sites (N = 31 vs. 17; NanoString cohort). D, High (above median) TIL-B densities by IHC were associated with better overall survival in TNBC (N = 15; Bart's IHC cohort), and in the basal-like subtype by high CD20 GEx (N = 241; KM plotter cohort; log rank test used to assess statistical significance). E, Kaplan–Meier survival curves display DMFS for naïve B cells, plasma cells, and memory B cells in TNBC (KCL GEx cohort) using CIBERSORT (20). Data were divided into four groups based on B lymphocyte subset and TIL levels stratified by semiquantitative TIL classification. Statistical significance was assessed using univariate Cox proportional hazards regression models. F, Representative IHC/IF images (Bart's IHC cohort) depicting nucleated cells (DAPI), epithelial cells (PanCK), B lymphocytes (CD20), and T lymphocytes (CD3) within TNBC TIL-B^low and TIL-B^high lesions. Scale bar, 50 μm. G, Representative TNBC (TIL-B^high) images highlighting numerous lymphoid aggregates (within white dash lines) consisting of B lymphocytes assembled adjacent to a T lymphocyte zone. Brown dash lines indicate carcinoma edge. Scale bar, 2 mm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 3.

Occurrence of B lymphocytes in stromal clusters. A, Representative IHC/IF images (Bart's IHC cohort) highlighting key TIL characteristics: clustered TIL versus dispersed TIL and stromal TIL (outside tumor nests) versus intratumoral TIL (within tumor nests). Scale bar, 50 μm. B, Quantitative assessment of TIL-B spatial and structural characteristics within TNBC (N = 15; left) intratumoral versus stromal; clustered versus dispersed (right). Patients ranked according to CD20⁺ TIL-B density (TNBC1 = highest), and overall survival data are indicated. Inset, patient samples split at median density into high and low TIL-B groups and the percentage of intratumoral (left) or clustered (right) B lymphocytes were analyzed. Statistical significance was determined using the Student t test. C, Characterization of TNBC TIL-B profile as stromal clustered, intratumoral clustered, stromal dispersed, or intratumoral dispersed. Overall survival data are indicated. D, GEx data for lymphoid assembly marker genes CXCL13 and DC-LAMP: normal breast versus TNBC (N = 10 vs. 131, KCL GEx cohort; left); non-TNBC vs. TNBC (N = 515 vs. 123, TCGA GEx cohort; middle); TNBC subtypes (N = 122, KCL GEx cohort; right). E, Survival analysis in KM Plotter of determined ER⁻HER2⁻/basal surrogate, HER2⁺, luminal A, and luminal B subtype KM plotter surrogate subgroups (KM plotter cohort; ref. 19). These indicate that expression of lymphoid cell assembly genes carries positive prognostic value in TNBC/basal-like and luminal B subtypes. Individual genes were evaluated in combination with each other gene (left) and gene set as a whole (right). ns, nonsignificant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Figure 4.

scRNA-seq analysis reveals BCR-driven TIL-B activatory signatures, and B lymphocyte functional trait gene markers predict positive survival outcome. A, UMAP visualization according to global GEx of single B lymphocytes pooled from the peripheral blood (1,476 cells) and tumors (1,021 cells) of eight patients (single-cell cohort), colored by relative normalized gene expression levels for CD20 and CD27. B, The detection of differentially expressed genes [DEG; on cells originally annotated by Azizi and colleagues (22) as B cells] demonstrates elevated expression of FOS, JUN, RGS1, and CD69, indicated by fold change (FC; determined using Wilcoxon rank sum test). C, Gene set enrichment analysis of TIL-B relative to circulating B lymphocytes using hallmark gene sets. Red, positive normalized enrichment scores (hallmark expression enhanced in TIL-B). D, Survival analysis in KM Plotter of determined ER⁻HER2⁻/basal surrogate, HER2⁺, luminal A, and luminal B subtype KM plotter surrogate subgroups (KM plotter cohort; ref. 19) for expression of gene signatures positively regulating key B lymphocyte properties (activation, proliferation, and differentiation). Representative genes listed for B lymphocyte proliferation (44 total in set). Signatures from all three functions carry positive prognostic value in the basal-like cancer subtype. Individual genes were evaluated in combination with each other gene (left) and gene set as a whole (right). E, CellPhoneDB (25) was applied to analyze B-cell–T-cell interactions (single-cell cohort). After FDR (FDR < 0.001) correction, communication pathways identified included lymphoid assembly, cytokine signaling, costimulation, T-cell–dependent B-cell activation, and cytotoxic T lymphocyte (CTL) activation. Circle sizes indicate P value, whereas color-coding represents the average expression level of interacting molecule 1 in cluster 1 and interacting molecule 2 in cluster 2. ****, P < 0.0001.

Figure 5.

Quantitative fluorescence IHC reveals elevated IgG⁺:IgA⁺ ratio within high TIL-B tumors, implicating expansion of IgG⁺ B lymphocytes within TNBC tumor microenvironment. A, Comparison of surface immunoglobulin-expressing B lymphocyte density in normal breast and TNBC with low TIL-B density (below median CD20⁺) and high TIL-B density (above median CD20⁺). B, Quantitative IHC analysis profiling the proportions of B lymphocytes present within the microenvironment of normal breast tissue (N = 10) and TNBC (N = 14) expressing each Ig isotype. C, Enumeration of IgG⁺:IgA⁺ B lymphocyte ratios. D, Right, example images illustrating IgG⁺ and IgA⁺ B lymphocytes in a typical TIL-B low individual and a TIL-B high individual. Automated cellular detection identifies IgA (green) and IgG (blue) B lymphocytes. Scale bar, 100 μm. E, Representative images depicting typical Ig isotype expression among B lymphocytes: IgM⁺ (red), IgA⁺ (green), and IgG⁺ (blue). Images from Bart's IHC cohort. Brown dash lines indicate margin of the cancer. White lines separate distinct regions of B lymphocyte compartments (PZ, plasma cell zone; F, B lymphocyte follicle). Scale bar, 100 μm. Statistical significance was determined using the Student t-test. ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6.

Single-cell RNA-seq data analysis reveals favor of IgG isotypes in TNBC, while isotype-switching gene markers predict positive survival outcome. A, UMAP visualization of B lymphocyte populations in non-TNBC versus TNBC (single-cell cohort). B, Percentage of each Ig isotype based upon raw data of Ig heavy-chain (Student t test). C, UMAP visualization for IgM and IgG1 isotypes colored by relative normalized gene expression levels (N = 1,021 cells). D, IgCH switch transcripts of single B lymphocytes (CD19⁺CD27⁺/CD20⁺CD27⁺/CD22⁺CD27⁺ single cells) were analyzed in non-TNBC and TNBC tissues and demonstrated more Ig isotype-switching events in the TNBC samples. E, Survival analysis in KM Plotter of determined ER⁻HER2⁻/basal surrogate, HER2⁺, luminal A, and luminal B subtype KM plotter surrogate subgroups (KM plotter cohort; ref. 19) for expression of gene signatures conferring isotype switching and those positively regulating isotype switching (IgG and IgA isotype switching). Signatures from all three functions carry positive prognostic value in basal-like cancer. Individual genes were evaluated in combination with each other gene (left) and the gene set as a whole (right). ns, nonsignificant; ***, P < 0.001; ****, P < 0.0001.

Figure 7.

B lymphocyte repertoire analyses of immunoglobulin isotype switching and clonal expansion in breast cancers. A, A total of 7,670 immunoglobulin heavy-chain sequences were analyzed (KCL sequencing cohort). Top 10 clones determined by B lymphocyte repertoire long read data analyses. Clonotypes were estimated via clustering CDR3 sequences. Top, bars depict sizes of clones and their breakdown by isotypes. Bottom, isotypes present in each clone are indicated by dots. Vertical lines signify co-occurrence of isotypes in the same clone. B, Clone size frequency distribution of IgM/IgA/IgG sequences in normal breast (1,771 sequences) and breast cancer (5,899 sequences). Kolmogorov–Smirnov analysis highlights significant differences in clone size frequency distributions. C, Mean sequences per clone of IgM/IgA/IgG isotypes. IgG and, to a lesser extent, IgA isotypes are clonally expanded, whereas on average, IgG isotypes have significantly larger clone sizes than IgA. D, Comparisons of IgA and IgG variable usage of V–J, D–J, and V–D genes extracted from normal breast, ER⁺ cancer, and TNBC. For each gene usage combination, dot size is proportional to the frequency before clonal expansion. Dot colors correspond to fold change in the number of sequences following clonal expansion, indicating the preference of B lymphocytes with that specific VDJ combination to be clonally expanded. E, Selection pressure in clonally related IgA and IgG. Clonally related sequences are represented as paired observations (gray lines), and selection pressure was considered separately for the complementarity determining regions and framework regions. Sequences are grouped into normal breast and breast cancer (containing two TNBC and two ER⁺ samples). Paired Wilcoxon tests were conducted, and P values were corrected (Benjamini–Hochberg) for multiple comparisons. ns, nonsignificant; *, P < 0.05; **, P < 0.01.