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B-cell-specific checkpoint molecules that regulate anti-tumour immunity
调节抗肿瘤免疫的 B 细胞特异性检查点分子

Received: 2 November 2021
收稿日期: 2021-11-02
Accepted: 17 May 2023
录用日期: 2023-05-17
Published online: 21 June 2023
网络出版日期: 2023-06-21
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Abstract 抽象

Lloyd Bod , Yoon-Chul Kye , Jingwen Shi , Elena Torlai Triglia , Alexandra Schnell , Johannes Fessler , Stephen M. Ostrowski , Max Y. Von-Franque , Juhi R. Kuchroo , Rocky M. Barilla , Sarah Zaghouani', Elena Christian , Toni Marie Delorey , Kanishka Mohib , Sheng Xiao', Nadine Slingerland , Christopher J. Giuliano , Orr Ashenberg , Zhaorong Li7 , David M. Rothstein , David E. Fisher , Orit Rozenblatt-Rosen , Arlene H. Sharpe , Francisco J. Quintana , Lionel Apetoh , Aviv Regev Vijay K. Kuchroo
劳埃德·博德 , 桂允哲 , 史静雯 , 埃琳娜·托莱·特里利亚 , 亚历山德拉·施内尔 , 约翰内斯·费斯勒 , 斯蒂芬·奥斯特罗夫斯基 , 马克斯·冯·弗兰克 , Juhi R. Kuchroo , 洛基·巴里拉 , 莎拉·扎古阿尼', 埃琳娜·克里斯蒂安 , 托妮·玛丽·德洛里 , 卡尼什卡·莫希布 , 盛晓, 娜丁·斯林格兰 , 克里斯托弗·朱利亚诺 , 奥尔·阿森伯格 , 李兆荣7 , 大卫·罗斯坦 , 大卫·费舍尔 , 奥里特·罗森布拉特-罗森 , 阿琳·夏普 , 弗朗西斯科·金塔纳 , 莱昂内尔·阿佩托 , 阿维夫·雷格夫· 维杰·库克鲁

Abstract 抽象

The role of cells in anti-tumour immunity is still debated and, accordingly, immunotherapies have focused on targeting and natural killer cells to inhibit tumour growth . Here, using high-throughput flow cytometry as well as bulk and single-cell RNA-sequencing and B-cell-receptor-sequencing analysis of B cells temporally during B16F10 melanoma growth, we identified a subset of B cells that expands specifically in the draining lymph node over time in tumour-bearing mice. The expanding B cell subset expresses the cell surface molecule cell immunoglobulin and mucin domain 1(TIM-1, encoded by Havcr1) and a unique transcriptional signature, including multiple co-inhibitory molecules such as PD-1, TIM-3, TIGIT and LAG-3. Although conditional deletion of these co-inhibitory molecules on B cells had little or no effect on tumour burden, selective deletion of Havcr1 in B cells both substantially inhibited tumour growth and enhanced effector cell responses. Loss of TIM-1 enhanced the type 1 interferon response in B cells, which augmented B cell activation and increased antigen presentation and co-stimulation, resulting in increased expansion of tumour-specific effector T cells. Our results demonstrate that manipulation of TIM-1-expressing cells enables engagement of the second arm of adaptive immunity to promote anti-tumour immunity and inhibit tumour growth.
细胞在抗肿瘤免疫中的作用仍然存在争议,因此,免疫疗法的重点是靶向 和自然杀伤细胞以抑制肿瘤生长 。在这里,使用高通量流式细胞术以及 B16F10 黑色素瘤生长期间 B 细胞的批量和单细胞 RNA 测序以及 B 细胞受体测序分析,我们鉴定了一个 B 细胞亚群,该亚群随着时间的推移在荷瘤小鼠的引流淋巴结中特异性扩增。扩增的 B 细胞亚群表达细胞表面分子 细胞免疫球蛋白和粘蛋白结构域 1(TIM-1,由 Havcr1 编码)和独特的转录特征,包括多种共抑制分子,如 PD-1、TIM-3、TIGIT 和 LAG-3。尽管 B 细胞上这些共抑制分子的条件性缺失对肿瘤负荷几乎没有影响,但 B 细胞中 Havcr1 的选择性缺失既显着抑制了肿瘤生长,又增强了效应 细胞反应。TIM-1 的缺失增强了 B 细胞中的 1 型干扰素反应,从而增强了 B 细胞活化并增加了抗原呈递和共刺激,导致肿瘤特异性效应 T 细胞的扩增增加。我们的结果表明,操纵表达 TIM-1 的细胞可以参与适应性免疫的第二臂,以促进抗肿瘤免疫并抑制肿瘤生长。

B cells have key roles in both innate and adaptive immunity. Distinct specialized cell subsets engage a range of responses from antigen presentation to antibody production and cells are one of the most abundant cell types of tumour-infiltrating leukocytes (TILs) , especially in melanoma . However, the role of B cells in anti-tumour immunity remains unclear. Here we examine the cell repertoire at the single-cell resolution from tumour-infiltrating cells and tumour-draining lymph nodes (dLNs) and identify and characterize a subset of B cells expressing the checkpoint molecule TIM-1. We find that targeting TIM-1 enables engagement of this cell subset, with subsequent enhancement of anti-tumour and cell responses and inhibition of tumour cell growth, with implications for approaches to cancer therapy.
B细胞在先天免疫和适应性免疫中都起着关键作用。不同的特化 细胞亚群参与从抗原呈递到抗体产生的一系列反应, 细胞是肿瘤浸润性白细胞 (TIL) 最丰富的细胞类型之一,尤其是在黑色素瘤 中。然而,B细胞在抗肿瘤免疫中的作用仍不清楚。在这里, 我们以单细胞分辨率检查肿瘤浸润 细胞和肿瘤引流淋巴结 (dLN) 的细胞库,并鉴定和表征表达检查点分子 TIM-1 的 B 细胞亚群。我们发现靶向 TIM-1 能够使该 细胞亚群参与,从而增强抗肿瘤 细胞反应并抑制肿瘤细胞生长,从而对癌症治疗方法产生影响。

Distinct cell infiltrates in B16F10 TME
B16F10 TME中的不同 细胞浸润

To understand the role of B cell subsets in regulating immune responses to tumours, we characterized B cells from tumours, and non-draining LNs (ndLNs) in the B16F10 melanoma mouse model. We confirmed that B cells infiltrate the tumour and are increased in frequency within the dLN compared with in the ndLN (Extended Data Fig. 1a). Depletion of B cells globally using anti-CD20 monoclonal antibodies significantly enhanced melanoma tumour growth; however, abrogating plasma cell generation (using mice) did not affect the tumour burden (Extended Data Fig. 1b,c). Tumour-infiltrating B cells had distinct expression profiles on the basis of bulk RNA-sequencing (RNA-seq) analysis compared with B cells from lymphoid tissues, reflecting the induction of proliferative and migratory pathways associated with B cell activation (Extended Data Fig. 1d-g). Moreover, tumour-infiltrating B cells were predominantly follicular B cells of the B2 lineage with bimodal IgD expression (Extended Data Fig. 1h). Thus, although plasma cells seemed to be dispensable, total cells produced an anti-tumour effect and displayed a distinct phenotype after infiltration in B16F10 tumours, prompting a deeper analysis.
为了了解 B 细胞亚群在调节对肿瘤的免疫反应中的作用,我们在 B16F10 黑色素瘤小鼠模型中表征了来自肿瘤的 B 细胞和非 引流 LN (ndLN)。我们证实,与ndLN相比,B细胞浸润肿瘤,并且dLN内的频率增加(扩展数据图1a)。使用抗 CD20 单克隆抗体在全球范围内耗竭 B 细胞显着增强了黑色素瘤肿瘤的生长;然而,消除浆细胞生成(使用 小鼠)不会影响肿瘤负荷(扩展数据图1b,c)。与来自淋巴组织的B细胞相比,基于大量RNA测序(RNA-seq)分析,肿瘤浸润B细胞具有不同的表达谱,反映了与B细胞活化相关的增殖和迁移途径的诱导(扩展数据图1d-g)。此外,肿瘤浸润性 B 细胞主要是 B2 谱系的滤泡 B 细胞,具有双峰 IgD 表达(扩展数据图 1h)。因此,尽管浆细胞似乎是可有可无的,但总 细胞在浸润B16F10肿瘤后产生了抗肿瘤作用并显示出明显的表型,这促使了更深入的分析。
C d b
h j Ref. 24 . 7 Ref. 20 Melanoma Ref. 21 Colorectal cancer
h j 参考文献 24 .7 参考文献 20 黑色素瘤 参考文献 21 结直肠癌

Fig. Characterization of cells expressing TIM-1 and several checkpoint molecules in mouse melanoma and human tumours. a, Workflow for singlecell transcriptome profiling of 34,071 viable leukocytes from TME, and ndLN samples. mice per time point (days 7 (D7; early), 10 (intermediate) and 16 (late)). s.c., subcutaneous. b, Uniform manifold approximation and projection (UMAP) embedding of all cells sequenced with each colour representing tissues of origin (left), timepoint (centre) and expression of Cd19 (right). c, UMAP visualization of the immune cell types. , conventional CD4 cells; cDC1/2/3, type 1, 2 and 3 conventional dendritic cells; NK, natural killer. d,e, UMAP visualization of the 6,226 B cells (dots) collected from wild-type mice bearing B16F10 melanoma, depicting tissues of origin (d) or Leiden cell clusters (resolution 0.85 , The frequencies of cells from each cluster within the tissues of origin (f) or from cluster 3 over time and tissues of origin (g).h, The -transformed fold change (FC) in RNA levels between B cells derived from cluster 3 with the rest of the clusters and between the and ndLN. , Bulk RNA-seq analysis of TIM-1 and TIM-1- B cells derived from dLNs and ndLNs of B16F10-bearing wild-type mice. . i, Pathway enrichment analysis of dLN-derived TIM-1+ B cells. FACS, fluorescence-activated cell sorting; FDR, false-discovery rate.j, The expression pattern of a set of selected genes. k,1, UMAP plot of published scRNA-seq dat depicting 2,615 B cells (dots) isolated from human tumours, coloured by cell clusters (k, left), selected gene expression (k, right) and immune checkpoint signature score (1,top), and a stacked bar graph displaying the frequencies of B cells derived from responder and pre- and post-ICB samples among each Leiden cluster (1, bottom).
无花果。 小鼠黑色素瘤和人类肿瘤中表达 TIM-1 和几种检查点分子的 细胞表征。a,对来自 TME 和 ndLN 样品的 34,071 个活白细胞进行单细胞转录组分析的工作流程。 每个时间点的小鼠(第7天(D7;早期),第10天(中间)和第16天(晚期))。皮下皮下注射。b,所有细胞的均匀流形近似和投影 (UMAP) 嵌入,每种颜色代表 Cd19 的起源组织(左)、时间点(中)和表达(右)。c,免疫细胞类型的UMAP可视化。 ,常规CD4 细胞;cDC1/2/3,1、2 和 3 型常规树突状细胞;NK,自然杀手。d,e,从携带 B16F10 黑色素瘤的野生型小鼠中收集的 6,226 个 B 细胞(点)的 UMAP 可视化,描绘了起源组织 (d) 或 Leiden 细胞簇(分辨率 0.85 ,来自起源组织 (f) 或来自簇 3 的每个簇的细胞随时间推移的频率和起源组织 (g).h,来自簇 3 的 B 细胞与其他簇之间的 RNA 水平的 转化倍数变化 (FC) 和 在 和 ndLN 之间。 ,来自携带 B16F10 的野生型小鼠的 dLN 和 ndLN 的 TIM-1 和 TIM-1- B 细胞的批量 RNA-seq 分析。 。i, dLN来源的TIM-1+ B细胞的通路富集分析。FACS,荧光激活细胞分选;FDR, false-discovery rate.j, 一组选定基因的表达模式。 k,1,已发表的 scRNA-seq dat 的 UMAP 图描绘了从人类肿瘤中分离出的 2,615 个 B 细胞(点),按细胞簇(k,左)、选定的基因表达(k,右)和免疫检查点特征评分(1,top)着色,以及显示每个 Leiden 簇中来自应答者和 ICB 前后样本的 B 细胞频率的堆叠条形图 (1, 底部)。

B16F10 tumour growth induces a specific cell subset
B16F10 肿瘤生长诱导特定 细胞亚群

To further decipher B cell heterogeneity, we performed 5' single-cell RNA-seq (scRNA-seq) combined with VDJ/B cell receptor (BCR)-seq (scRNA/BCR-seq) analysis of cells in the tumour microenvironment (TME), dLN and ndLN at three different timepoints of B16F10 melanoma growth (Fig. 1a,b and Extended Data Fig. 2). The 34,071 high-quality cell profiles were grouped by respective lineages and tissue origin, and expressed known marker genes, which we used for their annotation (Fig. 1c and Extended Data Fig. 2c). We searched for B cell populations that were expanded over time or in the three compartments (tumour, dLN and ndLN) on the basis of either transcriptional states or BCR clones (Fig. 1d and Extended Data Fig. 2d-h). Although known cell subset expression signatures and markers did not identify discrete B cell groups (except for germinal-centre-like B cells;Extended Data Fig. 2g), unsupervised graph clustering partitioned them into five distinct clusters (Fig. 1e and Extended Data Fig. 2h). The main separation was by tissue origin (Fig. 1f), with clusters 1 and 2 comprising tumour-infiltrating cells with a highly activated or inflammatory phenotype (Cd69, Cd86 or Cxcr4 in cluster 1; Cd274,Apoe or Hspa1a in
为了进一步破译 B 细胞异质性,我们在 B16F10 黑色素瘤生长的三个不同时间点对 肿瘤微环境 (TME)、dLN 和 ndLN 中的细胞进行了 5' 单细胞 RNA-seq (scRNA-seq) 联合 VDJ/B 细胞受体 (BCR)-seq (scRNA/BCR-seq) 分析(图 1a、b 和扩展数据图 2)。34,071 个高质量细胞图谱按各自的谱系和组织来源分组,并表达已知的标记基因,我们将其用于注释(图 1c 和扩展数据图 2c)。我们根据转录状态或BCR克隆(图1d和扩展数据图2d-h)搜索了随时间推移或在三个区室(肿瘤、dLN和ndLN)中扩增的B细胞群。尽管已知 的细胞亚群表达特征和标记物无法识别离散的 B 细胞群(生发中心样 B 细胞除外;扩展数据图2g),无监督图聚类将它们划分为五个不同的聚类(图1e和扩展数据图2h)。主要分离是按组织来源进行的(图 1f),簇 1 和簇 2 包括具有高度活化或炎症表型的肿瘤浸润 细胞(簇 1 中的 Cd69、Cd86 或 Cxcr4;Cd274,Apoe 或 Hspa1a 在

cluster 2), clusters 4 and 5 consisting of both dLN and ndLN B cells with a naive-like profile (Cr2, Cxcr5, Tnfrsf13c in cluster 4; Fcer2a, Tnfrsf13b in cluster 5) and cluster 3 mainly comprising cells from the tumour dLN with proliferative and germinal-centre-like profiles (Mki67,Aicda). The frequency of dLN cells in cluster 3 B cells augmented over time as tumours increased in size, suggesting a specific induction of cluster 3 in response to melanoma growth (Fig.1g), consistent with the expression of activation and germinal centre cell signatures in these cells. Moreover, BCR-based clonal analysis (using Immcantation) identified only a small fraction of cells expressing immunoglobulin heavy chain gamma (IGHG), and those cells were predominantly members of cluster 3 and were moderately clonally expanded within the dLN compartment (Extended Data Fig. .
簇 2)、簇 4 和 5 由具有幼稚样特征的 dLN 和 ndLN B 细胞组成(簇 4 中的 Cr2、Cxcr5、Tnfrsf13c;簇 5) 和簇 3 中的 Fcer2a、Tnfrsf13b 主要由来自肿瘤 dLN 的细胞组成,具有增殖和生发中心样特征 (Mki67,Aicda)。随着肿瘤大小的增加,簇 3 B 细胞中 dLN 细胞的频率随着时间的推移而增加,表明簇 3 响应黑色素瘤生长的特异性诱导(图 1g),与这些细胞中激活和生发中心 细胞特征的表达一致。此外,基于 BCR 的克隆分析(使用 Immcantation)仅鉴定出一小部分表达免疫球蛋白重链 γ (IGHG) 的细胞,这些细胞主要是簇 3 的成员,并且在 dLN 区室内适度克隆扩增(扩展数据图 1)。

TIM-1 marks checkpoint-expressing cells
TIM-1 标记表达 检查点的细胞

We sought to isolate and purify the cell subset that increases with tumour growth by identifying cell surface markers that are expressed on this B cell population. The dLN-derived expanded cluster 3 B cells expressed genes encoding specific cell surface markers, especially Haucr1, encoding TIM-1 (using ; Fig. 1h and Extended Data Fig. 2f). In the B16F10 tumour model, TIM-1 B cells poorly infiltrated the tumour but were found in the lymphoid organs and increased preferentially within the dLN (Extended Data Fig. 3a), consistent with our RNA profiles. TIM-1 is a member of the TIM family, of which TIM-3 is the most characterized molecule in the context of autoimmunity and anti-tumour immunity . TIM- 1 is not well studied in the context of cancer but is expressed on a fraction (around ) of peripheral B cells and can promote tissue tolerance by binding to phosphatidylserine exposed on apoptotic cells .
我们试图通过鉴定在该 B 细胞群上表达的细胞表面标志物来分离和纯化随着肿瘤生长而增加的 细胞亚群。dLN 衍生的扩增簇 3 B 细胞表达编码特定细胞表面标记物的基因,尤其是编码 TIM-1 的 Haucr1(使用 ;图1h和扩展数据图2f)。在 B16F10 肿瘤模型中,TIM-1 B 细胞浸润不良,但在淋巴器官中发现,并在 dLN 内优先增加(扩展数据图 3a),与我们的 RNA 图谱一致。TIM-1 是 TIM 家族的成员,其中 TIM-3 是自身免疫和抗肿瘤免疫背景下最具特征的分子 。TIM-1在癌症的背景下没有得到很好的研究,但在外周B细胞的一部分(周围 )上表达,并且可以通过与暴露在凋亡细胞 上的磷脂酰丝氨酸结合来促进组织耐受性。
Sorted TIM-1 and B cells from the dLN and ndLN of B16F10-bearing mice showed distinct transcriptional profiles on the basis of bulk RNA-seq and flow cytometry analysis (Fig. 1i,j and Extended Data Fig. 3b,c), clustering by TIM-1 expression and not tissue origin, with TIM- cells from the dLN displaying a unique expression signature, enriched in B cell activation and proliferation genes (Fig. li and Extended Data Fig. 3 b,c). These features of TIM- B cells were confirmed functionally in vitro, as TIM- cells had increased proliferation and differentiation into plasma cells (Extended Data Fig. 3d).
从携带 B16F10 的小鼠的 dLN 和 ndLN 中分选的 TIM-1 B 细胞在大量 RNA-seq 和流式细胞术分析的基础上显示出不同的转录谱(图 1i,j 和扩展数据图 3b,c),按 TIM-1 表达而不是组织来源聚类,来自 dLN 的 TIM- 细胞显示出独特的表达特征,富含 B 细胞活化和增殖基因(图 li 和扩展数据图 3 b,TIM-B 细胞的这些特征在体外功能上得到证实,因为TIM- 细胞的增殖和分化为浆细胞的增加(扩展数据Fig. 3d)。
However, scRNA-seq analysis of sorted TIM- and TIM-1 B cells from the , ndLN and spleen showed that germinal-centre-like TIM-1 B cellsconsist of only around of allTIM-1-expressingB cells(Extended Data Fig. 3e-g), indicating that TIM-1 is not simply a marker of germinal centres, or a unique B cell lineage. Instead, our data suggest that TIM-1 may be expressed on all cells during cell activation. Consistent with this model, TIM-1 is transiently induced across cell divisions on the cell surface of TIM-1 B cells after cell activation in vitro with and/or CD40 but not lipopolysaccharide (LPS), supporting that TIM-1 could be induced on all cells after antigen-driven cell activation (Extended Data Fig. ).
然而,对来自、ndLN和脾脏的TIM- 和TIM-1 B细胞进行scRNA-seq分析表明,生发中心样TIM-1 B细胞仅由所有表达TIM-1的B细胞组成 (扩展数据图3e-g),表明TIM-1不仅仅是生发中心的标志物或独特的B细胞谱系。 相反,我们的数据表明,TIM-1可能在细胞活化过程中 在所有 细胞上表达。与该模型一致,在体外用 和/或CD40而不是脂多糖(LPS) 激活细胞后,TIM-1在TIM-1 B细胞表面的细胞分裂中被瞬时诱导,支持TIM-1可以在抗原驱动的 细胞活化后在所有 细胞上诱导(扩展数据图1)。 )。
Notably, TIM- B cells from the dLN of B16F10 tumour-bearing mice also express higher levels of various co-inhibitory and immunoregulatory molecules that are expressed on T cells, including PD-1, TIGIT, LAG3, TIM-3, CD39, CD73 and IL-10 (Fig. 1j and Extended Data Fig. 4a,b). These molecules were preferentially induced on TIM cells compared with on TIM-1 B cells after treatment with anti-IgM or anti-CD40 antibodies or LPS stimulation in vitro (Extended Data Fig. 4c).
值得注意的是,来自B16F10荷瘤小鼠dLN的TIM-B 细胞也表达更高水平的T细胞上表达的各种共抑制和免疫调节分子,包括PD-1、TIGIT、LAG3、TIM-3、CD39、CD73和IL-10(图1j和扩展数据图4a,b)。在体外用抗 IgM 或抗 CD40 抗体或 LPS 刺激处理后,与 TIM-1 B 细胞相比,这些分子优先诱导在 TIM 细胞上(扩展数据图 4c)。
To study the relevance of TIM- cells in human tumours, we reanalysed TILs from human tumours using publicly available datasets that we and others have previously generated with high sensitivity (Smart-seq2 protocol) . While focusing on tumour-infiltrating cells derived from immune checkpoint blockade (ICB)-naive samples, we identified a cluster of B cells (cluster 4) co-expressing TIM-1 and multiple co-inhibitory molecules (HAVCR2, TIGIT,PDCD1,LAG3) and IL10, comprising a distinct cell subset and a signature that overlaps with human exhausted T cells (Fig.1k-l and Extended Data Fig. 4d,e).Notably, cells in cluster 4 , which largely included TIM- B cells, were more frequent among cells derived from ICB-naive patients and were decreased in TILs after checkpoint blockade therapy in human tumours (Fig. 1 l and Extended Data Fig. 4f,g). We corroborated these findings by investigating additional human cancer datasets derived from breast, colorectal, ovarian and lung tumours in which we could identify a similar cluster of B cells expressing checkpoint receptors (IC ) enriched in ICB-naive patient samples (Extended Data Fig. 4h-j). Clinically, high expression of HAVCR1 correlated with poor overall survival in patients with lung, pancreatic and stomach adenocarcinomas, while being protective in the context of colorectal cancer (Extended Data Fig. 4k,I). Furthermore, except for a poor impact on survival for stomach cancer, a high score for the cell signature did not affect the clinical outcomes of the patients (Extended Data Fig. ). These data indicate that TIM-1 marks a subset of activated cells expressing co-inhibitory molecules and IL-10 in both mouse and human tumours and their presence in human tumours seems to be inhibited after checkpoint blockade therapy.
为了研究 TIM- 细胞在人类肿瘤中的相关性,我们使用我们和其他人之前以高灵敏度生成的公开数据集(Smart-seq2 协议) 重新分析了来自人类肿瘤的 TIL。在关注源自免疫检查点阻断 (ICB) 初治样本的肿瘤浸润 细胞的同时,我们鉴定了一簇共表达 TIM-1 的 B 细胞(簇 4)和多个共抑制分子(HAVCR2、TIGIT、PDCD1、LAG3)和 IL10,包括一个不同的 细胞亚群和一个与人耗尽的 T 细胞 重叠的特征(图 1k-l 和扩展数据图 4d,e)。值得注意的是,簇 4 中的细胞(主要包括 TIM-B 细胞)在源自 ICB 初治患者的细胞中 更常见,并且在人类肿瘤中检查点阻断治疗后 TIL 中减少(图 1 l 和扩展数据图 4f,g)。我们通过研究来自乳腺癌、结直肠癌、卵巢癌和肺肿瘤的其他人类癌症数据集来证实这些发现,在这些数据集中,我们可以识别出一个类似的 B 细胞簇,这些细胞表达富含 ICB 初治患者样本中的检查点受体 (IC )(扩展数据图 4h-j)。临床上,HAVCR1 的高表达与肺癌、胰腺癌和胃腺癌患者的总生存期差相关,同时在结直肠癌的背景下具有保护作用(扩展数据图 4k,I)。此外,除了对胃癌生存率的影响较差外, 细胞特征的高分不会影响患者的临床结果(扩展数据图1)。 )。 这些数据表明,TIM-1 在小鼠和人类肿瘤中都标记了表达共抑制分子和 IL-10 的活化 细胞子集,并且在检查点阻断治疗后,它们在人类肿瘤中的存在似乎受到抑制。

Genetic deletion of TIM-1 in B cells limits tumour growth
B 细胞中 TIM-1 的基因缺失限制了肿瘤的生长

As TIM- B cells expressed multiple known cell checkpoint molecules, some previously reported in B cells , we investigated their B-cell-intrinsic roles in regulating anti-tumour immunity. Conditional deletion of the checkpoint molecules Havcr2, Tigit, Pdcd1 (encoding PD-1) or Lag3 in B cells had a modest impact or no effect on tumour growth (Fig. 2a-e). Only loss of TIGIT on B cells led to a modest but significant decrease in tumour growth. Although IL-10 has previously been associated with regulatory cells and shown to be a critical driver of B cell regulatory function , loss of B-cell-specific IL-10 had no effect on B16F10 growth, arguing against a functional role of IL-10-producing B cells in this melanoma model (Fig. 2f).
由于 TIM-B 细胞表达了多种已知 的细胞检查点分子,其中一些先前在 B 细胞 中报道过,我们研究了它们在调节抗肿瘤免疫方面的 B 细胞内在作用。B细胞中检查点分子Havcr2、Tigit、Pdcd1(编码PD-1)或Lag3的条件性缺失对肿瘤生长有适度影响或没有影响(图2a-e)。只有 B 细胞上 TIGIT 的缺失导致肿瘤生长适度但显着减少。尽管 IL-10 以前与调节 细胞 相关并被证明是 B 细胞调节功能 的关键驱动因素,但 B 细胞特异性 IL-10 的缺失对 B16F10 的生长没有影响,反对产生 IL-10 的 B 细胞在该黑色素瘤模型中的功能作用(图 2f)。
Conversely, conditional deletion of Havcr1 on B cells substantially inhibited tumour growth in various B16F10 melanoma tumour models, as well as MC38 colon carcinoma or KP1.9 lung adenocarcinoma (Fig. 2g-i and Extended Data Fig.5a-e), indicating that TIM-1 is not only a marker of checkpoint-receptor-expressing B cells, but that TIM-1 has a functional role in regulating tumour growth in vivo. Notably, although TIM-1 was initially described to be expressed on T cells, Havcr1 conditional deletion using , which deleted TIM-1 on all T cells, had no effect on tumour growth in mice implanted with B16F10 melanoma (Extended Data Fig. 5f,g), supporting a cell-intrinsic role of TIM-1 in B cell function. Together, these data demonstrate an important role of TIM-1 specifically expressed on B cells in regulating anti-tumour immune responses and tumour growth in vivo.
相反,在各种 B16F10 黑色素瘤肿瘤模型以及 MC38 结肠癌或 KP1.9 肺腺癌中,B 细胞上 Havcr1 的条件性缺失显着抑制了肿瘤生长(图 2g-i 和扩展数据图 5a-e),表明 TIM-1 不仅是表达检查点受体的 B 细胞的标志物,而且 TIM-1 在体内调节肿瘤生长中具有功能作用。值得注意的是,尽管 TIM-1 最初被描述为在 T 细胞上表达,但使用 Havcr1 条件性缺失在所有 T 细胞上删除 TIM-1,对植入 B16F10 黑色素瘤的小鼠的肿瘤生长没有影响(扩展数据图 5f,g),支持 TIM-1 在 B 细胞功能中的细胞内在作用。总之,这些数据表明,在B细胞上特异性表达的TIM-1在调节体内抗肿瘤免疫反应和肿瘤生长方面具有重要作用。

Therapeutic targeting of TIM-1 reduces tumour growth
TIM-1 的治疗靶向可减少肿瘤生长

To examine whether acute deletion of Havcr1 also regulates tumour growth, we generated CD20.TamCre Havcr (hereafter, Havcri ) mice and treated the mice with tamoxifen to trigger acute Cre-mediated Havcr1 deletion and observed inhibition of tumour growth similar to that with constitutive deletion of Havcr1 in B cells (Extended Data Fig. 5h).Moreover, this indicates that deletion of TIM-1 on B cells using another Cre driver independent of induces similar control of tumour growth.
为了检查 Havcr1 的急性缺失是否也调节肿瘤生长,我们生成 了 CD20。TamCre Havcr (以下简称Havcri )小鼠并用他莫昔芬处理小鼠以触发急性Cre介导的Havcr1缺失,并观察到肿瘤生长的抑制类似于B细胞中Havcr1的组成型缺失(扩展数据图5h)。此外,这表明使用另一个独立于 Cre 的驱动因素在 B 细胞上缺失 TIM-1 会诱导类似的肿瘤生长控制。
Next, therapeutic administration of a commercially available high-affinity anti-TIM-1 antibody (3B3) also induced marked inhibition of B16F10 tumour growth (Extended Data Fig. 5i). This therapeutic effect required the presence of B cells, and of TIM-1 expression on B cells, such that the therapeutic effect of the anti-TIM-1 antibody was lost in MT (lacking B cells) or Havcr mice (Fig. 3a and Extended Data Fig. 5i,j). Notably, we found that anti-TIM-1 treatment had a therapeutic effect inhibiting tumour growth selectively in mice with intact MHCII
接下来,市售高亲和力抗 TIM-1 抗体 (3B3) 的治疗性给药也诱导了对 B16F10 肿瘤生长的显着抑制(扩展数据图 5i)。这种治疗效果需要B细胞的存在,以及B细胞上TIM-1的表达,使得抗TIM-1抗体的治疗效果在MT(缺乏B细胞)或Havcr 小鼠中 丧失(图3a和扩展数据图5i,j)。值得注意的是,我们发现抗 TIM-1 治疗具有治疗作用,可选择性地抑制完整 MHCII 小鼠的肿瘤生长

h
Fig. 2 | Screening of in vivo regulatory molecules reveals TIM-1 as a B cell immune checkpoint controlling tumour growth. a-f, Subcutaneous (s.c.) B16F10 melanoma growth in Cd19 , Tigit (c), and controls versus mice. a, Experimental schematic. g-i, Schematic (g), quantification (h) and imaging (i) of tumour growth in and mice implanted s.c. with ( control versus Havcr ) or intravenously (i.v.) injected with KP1.9 cells ( mice per group). Tumour burden was assessed by histological analysis of lung tissue collected 4 weeks after injection. Data are mean s.e.m. and pooled from two to three independent experiments. Statistical analysis was performed using repeated-measures two-way analysis of variance (ANOVA) (b-f and ) and two-tailed Student's -tests (i). Scale bar, (i). expression on the B cell surface (Extended Data Fig. ). Whereas 3B3 has previously been reported to be an agonistic antibody based on activating cell effector functions, in B cells, the effects of the 3B3 antibody are very similar to what we observed after the genetic loss of TIM-1 on B cells. Whether this is due to differential effects of TIM-1 on T cells versus cells needs to be further characterized; nonetheless, the therapeutic effects of anti-TIM-1 antibodies on tumour growth are unequivocal. As TIM-1 expression on T cells has no effect on tumour growth, in vivo effects of anti-TIM-1 antibodies appear to be entirely dependent on the expression of TIM-1 on B cells. Moreover, we performed anti-TIM-1 treatment experiments using the spontaneous melanoma
图2 |体内调节分子的筛选显示 TIM-1 是控制肿瘤生长的 B 细胞免疫检查点。a-f,皮下 (sc) B16F10 黑色素瘤在 Cd19 中的生长,Tigit (c) 和对 照组与 小鼠的对比。a, 实验示意图。g-i,示意图(g),定量(h)和成像(i)肿瘤生长 植入皮下注射 对照与 Havcr )或静脉注射(i.v.)注射KP1.9细胞(每组 小鼠)的小鼠。通过对注射后 4 周收集的肺组织进行组织学分析来评估肿瘤负荷。数据是平均 的,并汇集了两到三个独立的实验。使用重复测量双因素方差分析(ANOVA)(b-f和 )和双尾学生 检验(i)进行统计分析。比例尺, (i)。在B细胞表面的表达(扩展数据图。 )。虽然 3B3 之前被报道为一种基于激活 细胞效应功能的激动性抗体,但在 B 细胞中,3B3 抗体的作用与我们在 B 细胞上 TIM-1 遗传缺失后观察到的非常相似。这是否是由于 TIM-1 对 T 细胞与 细胞的不同影响需要进一步表征;尽管如此,抗 TIM-1 抗体对肿瘤生长的治疗作用是明确的。由于 TIM-1 在 T 细胞上的表达对肿瘤生长没有影响,因此抗 TIM-1 抗体的体内效应似乎完全依赖于 TIM-1 在 B 细胞上的表达。此外,我们使用自发性黑色素瘤进行了抗 TIM-1 治疗实验

ing a tamoxifen-inducible Cre-recombinase under the control of the tyrosinase promoter. This model enables melanocyte lineage-specific induction of a BRAF(V600E) mutation and deletion of Pten, inducing spontaneous formation of melanoma and replicating many of the features of human melanoma. Notably, treatment with anti-TIM-1 (clone 3B3) significantly reduced melanoma genesis and proximal metastatic dissemination (Fig. 3b,c). Finally, combined PD-1blockade (as a T-cell-relevant target) together with anti-TIM-1 antibody treatment had an additive effect, consistent with an impact on two different compartments, resulting in more rapid and consistent growth control and prolonged survival in B16F10-bearing mice compared with either treatment alone (Fig. 3d and Extended Data Fig.5I).Monotherapy with anti-TIM-1 antibodies or in combination with PD-1blockade was accompanied by an increased frequency of effector and cells infiltrating the tumours of antibody-treated animals, without affecting B cell or regulatory cell infiltration (Extended Data Fig. ) and with an induction of a larger fraction of granzyme cells and cells among both the CD4 and cell compartments (Fig. 3e and Extended Data Fig. 5n). Together, these data show that therapeutic antibody blockade of TIM-1 in vivo results in tumour growth control of both transplanted and spontaneous tumour models and requires TIM-1 expression on B cells, but not on other cell types, which is consistent with the phenotype observed in tumour-bearing mice with genetic deletion of Havcr1 in B cells.
在酪氨酸酶启动子的控制下,他莫昔芬诱导的Cre-重组酶。该模型能够对 BRAF(V600E) 突变和 Pten 缺失进行黑色素细胞谱系特异性诱导,诱导黑色素瘤的自发形成并复制人类黑色素瘤的许多特征。值得注意的是,抗 TIM-1(克隆 3B3)治疗显着减少了黑色素瘤的发生和近端转移播散(图 3b、c)。最后,PD-1阻断剂(作为T细胞相关靶标)与抗TIM-1抗体治疗联合使用具有累加效应,与对两个不同区室的影响一致,与单独治疗相比,在携带B16F10的小鼠中,生长控制更快,更一致,生存期更长(Fig. 3d和扩展数据图5I)。抗 TIM-1 抗体单药治疗或与 PD-1 阻断剂联合使用时,效应器 细胞浸润抗体治疗动物肿瘤的频率增加,而不影响 B 细胞或调节 细胞浸润(扩展数据图 1)。 ),并在CD4 细胞区室中诱导更大比例的颗粒酶 细胞和 细胞(图3e和扩展数据图5n)。总之,这些数据表明,体内 TIM-1 的治疗性抗体阻断导致移植和自发性肿瘤模型的肿瘤生长控制,并且需要 TIM-1 在 B 细胞上表达,但在其他细胞类型上不表达,这与在 B 细胞中 Havcr1 基因缺失的荷瘤小鼠中观察到的表型一致。

Loss of TIM- 1 in B cells enhances effector T cell responses
B 细胞中 TIM-1 的缺失增强了效应 T 细胞的反应

To investigate how TIM-1 loss in B cells affects tumour growth, we analysed the composition of cells in the TME, dLN and ndLN of control or Havcr mice using flow cytometry at 16 days after receiving subcutaneous B16F10 cells (Fig. 4a,b and Extended Data Fig. 6). There was an increased immune cell infiltration in Havcr tumours versus control tumours (Extended Data Fig. 6b), and a significant increase in
为了研究 B 细胞中 TIM-1 缺失如何影响肿瘤生长,我们在接受皮下 B16F10 细胞后 16 天使用流式细胞术分析了对照组或 Havcr 小鼠的 TME、dLN 和 ndLN 中的细胞组成(图 4a、b 和扩展数据图 6)。与对照组相比,Havcr 肿瘤中的免疫细胞浸润增加(扩展数据图6b),并且

d
Fig. 3 | Targeting of TIM-1 reduces B16F10 growth, is dependent on TIM-1 expression on B cells and augments PD-1 blockade therapy. a, B16F10 tumour growth in and mice ( mice per group) that were treated with anti-TIM-1 or isotype control antibodies.b,c,Braf-Pten mice were painted with 4-hydroxytamoxifen (tamox.) on one ear and treated with anti-TIM-1 antibodies beginning 27 days later when visible lesions were apparent. Representative photographs, and measurements of pigmentation (b) and the number of facial nodules (c) are shown for isotype-treated ( mice) or
图3 |靶向 TIM-1 可减少 B16F10 的生长,依赖于 B 细胞上 TIM-1 的表达并增强 PD-1 阻断治疗。a,B16F10肿瘤生长和 用抗TIM-1或同型对照抗体治疗的小鼠(每组 小鼠).b,c,Braf-Pten小鼠在一只耳朵上涂上4-羟基他莫昔芬(tamox.),并在27天后开始用抗TIM-1抗体治疗,当可见病变明显时。代表性照片,色素沉着(b)和面部结节(c)的测量值显示为同型处理( 小鼠)或
initiation/7 weeks after tumour induction. Data are mean s.e.m. pooled from anti-TIM-1-treated ( mice) ears at treatment and 3 weeks after treatment two to three independent experiments. d,e, Tumour growth (d) and flow cytometry immunophenotyping of TILs showing the frequencies of IFN cells among and TILs (e) of C57Bl/6J mice implanted with B16F10 melanoma and treated with anti-TIM-1, anti-PD-1, anti-TIM-1+ anti-PD-1 (combo) or isotype controls. mice per group for tumour growth analysis and mice per group for flow cytometry analysis. Statistical analysis was performed using repeated-measures two-way ANOVA ( and d) and one-way ANOVA with Tukey's multiple-comparison test (e).
开始/肿瘤诱导后 7 周。数据是在治疗时和治疗后 3 周的两到三个独立实验中从抗 TIM-1 治疗( 小鼠)耳朵汇总的平均 s.e.m. 合并的。d,e,TIL的肿瘤生长(d)和流式细胞术免疫表型,显示植入B16F10黑色素瘤的C57Bl/6J小鼠的IFN 细胞频率 TILs(e),并用抗TIM-1,抗PD-1,抗TIM-1+抗PD-1(组合)或同型对照治疗。 每组小鼠进行肿瘤生长分析, 每组小鼠进行流式细胞术分析。使用重复测量的双因素方差分析( 和d)和单因素方差分析以及Tukey的多重比较检验(e)进行统计分析。
the frequency of cells, and decreased frequency of cells ( cells) among cells, resulting in an approximately fourfold increase in the ratio of cells to cells (Extended Data Fig. . Moreover, there was a decreased proportion of cells within the dLN of Havcr mice (Extended Data Fig. ). Myeloid cell subsets and cells were unchanged in either the tumour or the LNs (Extended Data Fig. 6f). Moreover, among TILsfrom Havcr mice, a larger fraction of and cells secreted both TNF and IFN in tumours compared with the control mice, and cells displayed a stronger cytotoxic profile, with elevated expression of CD107a and an increased frequency of cells co-expressing granzyme and perforin or the transcription factors EOMES and TBET that regulate IFN production (Fig. 4a,b and Extended Data Fig. 6f,g). However, IL-2 production was not changed in or cells (Fig. 4 a), and there were no alterations in TCF1 expression levels or in the co-expression of the checkpoint molecules PD-1 and TIM-3 (Extended Data Fig. 6h,i). Similar results were obtained in mice that received MC38 colon adenocarcinoma (Extended Data Fig. 61).
细胞的 频率,以及细胞之间 细胞( 细胞)频率的降低,导致 细胞与 细胞的比例增加约四倍(扩展数据图1)。 。此外,Havcr 小鼠dLN内 的细胞比例降低(扩展数据图1)。 )。肿瘤或LN中的髓系细胞亚群和 细胞均未改变(扩展数据图6f)。此外,在来自Havcr 小鼠的TIL中,与对照小鼠相比,肿瘤中分泌TNF和IFN 细胞比例更大 ,并且 细胞表现出更强的细胞毒性特征,CD107a的表达升高,共表达颗粒酶 和穿孔素或调节IFN 的转录因子EOMES和TBET的 细胞频率增加生产(图4a,b和扩展数据图6f,g)。然而,IL-2 的产生在细胞 中没有 改变(图 4 a),并且 TCF1 表达水平或检查点分子 PD-1 和 TIM-3 的共表达没有改变(扩展数据图 6h,i)。在接受MC38结肠腺癌的小鼠中也获得了类似的结果(扩展数据图61)。
To further characterize these changes in the tumours of Havcr1 mice, we profiled cells infiltrating the tumours, and ndLN from these mice by combined single-cell RNA- and TCR-seq (scRNA/TCR-seq; Fig. 4c,d and Extended Data Fig. 7a,b). scRNA-seq confirmed an increase in cytotoxic cell infiltration in Havcr tumours versus the controls and showed a higher frequency of clonally expanded CD8 cells in Havcr1 tumours on the basis of TCR analysis ( versus of clones with more than 2 cells) (Fig. 4e,
为了进一步表征 Havcr1 小鼠肿瘤的这些变化,我们 通过联合单细胞 RNA- 和 TCR-seq (scRNA/TCR-seq;图4c,d和扩展数据图7a,b)。scRNA-seq证实,与对照组相比,Havcr 肿瘤中细胞毒性 细胞浸润增加,并且根据TCR分析( 与具有2个以上细胞的克隆相比 ),Havcr1 肿瘤中克隆扩增的CD8 细胞的频率更高(图4e,

Methods and Extended Data Fig. 7c). Notably, clonally expanded CD8 cells from tumours displayed a higher expression of genes associated with an effector/cytotoxic phenotype (that is, Gzmb, Gzma, Gzmc, Prf1, Ifng and Ccl4) (Fig. 4f,g and Extended Data Fig. 7d). Consistently, TILs from B16-OVA-bearing mice showed an increased frequency of proliferating OVA-specific cells in Havcr tumours versus the control as determined by dextramer staining and Ki-67 expression (Fig. 4h). Taken together, these data indicate that the deletion of Havcr1 in B cells resulted in decreased infiltration and increased clonally expanded antigen-specific TILS.
方法和扩展数据图7c)。值得注意的是,来自 肿瘤的克隆扩增的CD8 细胞显示出与效应/细胞毒性表型(即Gzmb,Gzma,Gzmc,Prf1,Ifng和Ccl4)相关的基因的更高表达(图4f,g和扩展数据图7d)。一致地,来自携带 B16-OVA 的小鼠的 TIL 显示 Havcr 肿瘤中 OVA 特异性 细胞增殖的频率增加,与通过