Humoral IgG1 Response Predicts Immunotherapy Outcomes

PCs are enriched in clinical responders to PD-1 blockade

To test the hypothesis that ⁢B cells or PCs have a key role during immunotherapy and ⁤treatment response, we examined specimens from eight sets of self-reliant clinical trial cohorts: two newly generated datasets from our own investigator-initiated trials in patients with HCC treated with neoadjuvant PD-1 blockade, with or⁢ without‍ radiation, for discovery and validation, respectively. We also analyzed published datasets of a combination of PD-1, PD-L1, CTLA-4 and VEGF-A blockade, including large cohorts such as IMbrave150 and The Cancer Genome Atlas (TCGA) (Fig. 1a). No sex-associated differences were observed in any of the analyzed outcomes, consistent with the balanced ⁢depiction of male and⁤ female ⁤patients in the cohort.

JCHAIN (Fig. 1c). Naive B cells dominated ⁣the B cell fraction of the immune cells⁣ across the tissue compartments, followed by memory B cells, relative to total B cells and PCs (Extended Data Fig. 1c-f).As expected, canonical and cluster-specific genes were expressed⁣ in more ⁢than ⁣70% of cells (Extended Data Fig. 1g,h). Pathological responses to neoadjuvant PD-1 were defined as more than⁢ 50% necrosis of tumor at the time of surgery, followed‍ by differential cell abundance showing an enrichment of all PC phenotypes in the tumor among ICB responders (FDR < 0.05), which was not as pronounced in adjacent uninvolved liver (Fig. 1d,e). Conversely, non-responders were enriched in unswitched memory B cells in tumor (FDR < 0.05) but less so in normal tissues (Fig. 1d,e).

Preexisting IgG1 PCs associated with clinical response are expanded after PD-1 blockade

We⁣ hypothesized that ⁢skewing of IgG1-producing PCs is linked ‍to clinical response, given their overrepresentation in single-cell data from ‍responders (extended Data Fig. 1i). To test this, we used complementary bulk RNA-seq of paired pretreatment biopsies and posttreatment resected tumors from⁤ the discovery cohort. Principal component analysis showed that gene expression profiles were markedly ⁤diffrent between responders and non-responders (Fig.1f,g and⁢ Extended⁢ Data Fig.1j-k).

In posttreatment tumors, IGHG1 emerged as one of the top upregulated genes. Its expression was already elevated in pretreatment samples‍ from responders and increased significantly after therapy, whereas ⁤it remained unchanged or decreased⁤ in non-responders (FDR < 0.01; Fig. ⁢ 1f,g ⁢and Extended data Fig. 1j-k).

a, Flowchart illustrating Ig isotype mapping for 37,000 single B⁤ cells, including 30,000 cells with paired BCR-seq and 7,000 additional cells mapped via gene-expression-based inference. Bottom panels show correlations between isotype ⁣assignments from gene expression and scBCR-seq⁤ for the 30,000 cells with paired data. b, Stacked bar plots showing isotype composition per sample and per patient. Multiple samples per patient were sequenced to ensure reproducibility. Single-cell BCR-seq data include six patients with HCC‍ treated with ICB (two responders and four non-responders). Using scRNA-seq-based isotype inference, the analysis was expanded to 8 responders and 14 non-responders. The y axis represents the proportion of cells (0-1). Consistent across BCR-seq and scRNA-seq-rescued⁢ isotypes, PC clusters are enriched for IgG1/IgG2, whereas naive and memory B cell clusters predominantly express IGHM and ⁢ IGHA (bottom bar plots and pie charts). c, Volcano plot ‍showing Dirichlet regression comparing responder and non-responder frequencies across clusters and isotypes ⁤(from both scRNA-seq and scBCR-seq). The log-transformed fold changes⁢ and P values were obtained from log-likelihood tests;⁢ adjusted P values were computed using Benjamini−Hochberg⁢ correction. PCs and IgG1/IgG2 responses are significantly enriched in responders. d, Box plot (as defined in Fig. 1c) showing increased IgG1⁺ ‍ PC representation in tumor tissue of responders ⁣with available BCR-seq. Ratios were estimated using Dirichlet regression with log-likelihood testing. e, Stacked bar plot of expanded clones per cluster, defining expansion as two or more cells per clonotype.

We examined the spatial distribution of the B cells (CD20⁺), PCs (MZB1⁺) and other immune cells in the tumor and adjacent liver in 17 mIHC biopsies (6 responders and 11 non-responders) from our discovery cohort. We observed that among anti-PD-1⁢ responders, the MZB1⁺ PCs were highly infiltrative⁢ throughout the tumor parenchyma compared to the PCs in non-responders (Fig. ⁤3a,b). Next,we used a radial binning approach to define cell communities or immune aggregates in an unsupervised fashion (Fig. 3c). ⁢Hear, responders showed enrichment of CD3⁺CD8⁺ T cells, CD68⁺ ⁣ macrophages and MZB1⁺ PCs. Conversely,CD20⁺ B cells were found ‍within the lymphoid aggregates or within the stromal compartment of the tumor rather than ⁤admixed with tumor parenchyma,reinforcing PC expansion as a hallmark of effective ICB response (Fig. 3d).

Fig. ‍3: Spatial analysis of immune infiltrating cells.a, Representative examples showing increased⁣ PC infiltration in responders compared to non-responders, quantified as the percentage of PCs within unsupervised neighborhood‍ regions derived from⁣ mIHC images. P, patient.

In ICB responders, we saw a marked enrichment of‍ IgG1⁺ PCs confined to an immune-rich T cell/B cell/vascular fibroblast hybrid niche,⁢ with significant upregulation of IL-4 and IL-13 signaling (Fig. 3e,f). ‍In⁤ ICB responders, we saw a marked enrichment of IgG1⁺ pcs confined to ⁣an immune-rich T ‍cell/B cell/vascular fibroblast hybrid ⁤niche, with significant upregulation of IL-4 and IL-13 signaling (Fig.3g,h). Pathway analysis of these clusters revealed a pro-BCR/TCR signaling⁣ hub, marked by MZB1,⁤ IL2RG, FCER2, PRDM1 ‍ and CD79A, consistent with a highly ⁢immunogenic, PC-driven microenvironment (Fig.3h,i). by contrast, non-responders harbored focal accumulations of CD27⁺ memory and dysfunctional B cells within ‍fibroinflammatory stromal regions. These niches were accompanied by regulatory T,T helper⁢ 17 (T_H17) and monocyte signatures,collectively defining an immunosuppressive ‘stromal memory/fatigued B cell reservoir’ (fig. 3g-j).

IgG1 PCs in tumor and lymph nodes increase after radiation therapy and PD-1 checkpoint⁣ blockade

To validate these findings in an independent cohort¹⁸,we analyzed B cells and PCs from patients with HCC treated with neoadjuvant stereotactic radiation followed ‍by anti-PD-1 therapy (biopsy,lymph node,tumor and⁢ adjacent normal) (Fig.1a and⁢ Extended Data ⁢Fig. 3a-c). In bulk sequencing, differential gene expression between responders and non-responders was most⁣ pronounced in pretreatment biopsies and posttreatment tumors but minimal in lymph nodes and adjacent normal ⁤tissues (Extended Data Fig. 3d-h). After treatment, total immunoglobulin increased, with responders showing selective IgG1 enrichment⁤ (Extended Data ⁤Fig. <

V2 cohort (radiation + anti-PD-1): a,b, Differential expression analysis of adjacent normal and tumor tissues revealed IGHG1 as⁣ the ‍most significant gene associated with response, using a two-sided moderated t*-test. c, Tumor heavy-chain isotype composition by cell cluster ⁤showed *IGHG1 enrichment in responders.⁤ d, Differential abundance⁣ model.

PCs of responders (Fig. 4i-k and Extended Data Fig.5e). Next, we validated our findings in advanced HCC treated with anti-PD-1 and anti-VEGF-A therapies in two independent cohorts of unresectable HCC: IMbrave150 (ref. ²⁰) and Cappuyns et al.²¹ (Fig. 4l,m).In both datasets, responders exhibited a⁤ skewing toward⁢ the IgG1 isotype, with ‍IgG1 emerging as the dominant subclass. Another independent cohort of patients with HCC and intrahepatic cholangiocarcinoma⁤ (ICCA)²² showed that HCC tumors had a low tumor diversity score, linked to favorable outcomes, whereas ICCA ⁢tumors exhibited a high tumor diversity score, linked to a more aggressive phenotype and worse outcomes (6-month survival⁤ for ICCA versus 26-month survival ⁢for HCC).Notably, ‍HCC samples had⁢ greater PC abundance, consistent with better progression-free survival in HCC than in ⁣ICCA (Fig. 4n,o). Together, these findings suggest that IgG1 PCs⁤ are⁤ strongly linked to ICB ⁤response.

ICB responders produce IgG antibodies‍ against cancer antigens

Given the enrichment ‍of IgG1 PCs in ICB responders, we explored whether ⁣theseb, Pie chart and bar plots showing that antibodies against cancer-associated ⁤antigens are predominantly enriched in responders compared to non-responders, respectively.c,d, ELISpot analysis⁢ of IFNγ-secreting cells in response to varying effector-to-target (E:T) ratios. The left panel shows wells with decreasing numbers of spots from top to bottom, corresponding to different ratios (1:1 and 5:1) for two conditions, indicating the frequency of cytokine-producing cells. The right ⁢panels represent different experimental conditions or treatments, with each row representing different⁣ replicates or conditions. Darker⁣ and more numerous spots indicate⁢ higher frequencies of cells secreting‍ IFNγ. e, Similarly, a broader⁢ characterization using seromics indicates increased abundance of autoantibodies against CTAs in responders compared to non-responders. f, Number of ⁤antigens enriched between‍ responders and ‍non-responders. g,⁤ Analysis of 16 patients (8 responders⁤ and⁣ 8 non-responders) showed that an enrichment of antibodies against CTA, tumor-associated antigen ⁤(Tu/AutoAg) ‍and other antigens was also higher ⁣in ⁣responders. Specifically, comparisons of CTA-specific IgG and ⁤IgA levels between responders and non-responders showed statistically significant differences ⁢(P = 0.04 and P = 0.01, ⁣respectively), as persistent by the Wilcoxon rank-sum test and visualized by box plots (same definition as in⁣ Fig. 1c). h, ⁣Box plots⁤ (same definition as in Fig. 1c) showing the CTA gene expression signature divided by timepoint (pre or post) and clinical response (responders and ⁢non-responders);‍ paired t-test and Wilcoxon rank test were used to estimate the ‍significance between both groups; only paired t-test results are shown in the figure. i,j, The increase⁢ in autoantibodies ⁣against CTAs in responders, specifically in the IgG1 and‍ IgA isotypes, was assessed using a heatmap⁢ to visualize relative abundance patterns across samples, violin plots to display the distribution and ⁢variability of antibody levels and quantile−quantile (Q−Q)⁢ plots to evaluate deviations from normality and highlight differences in distribution between responders and non-responders.KS, Kolmogorov–Smirnov test; NS, not significant; P/I, phorbol myristate acetate (PMA) and ionomycin positive control.