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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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 和 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。

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）。

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）。 。

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 和 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。

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。

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 和 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 和 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 和 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。

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 和 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 的 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 右聚糖染色和 Ki-67 表达确定的对照组相比（图 4h）。综上所述，这些数据表明，B细胞中Havcr1的缺失导致浸润减少 和克隆扩增抗原特异性 TILS增加。

To determine the mechanism by which Havcr1 deletion in B cells influenced T-cell-mediated anti-tumour responses, we analysed the B-cell-intrinsic effects of the genetic loss of Havcr1. Although there were no differences in the total frequency of B cells in Havcr tumours, dLNs and ndLNs relative to their respective controls (Extended Data Fig. 8a), scRNA-seq profiles of Havcr1 cells from dLNs and tumours (but not ndLNs) had a higher expression of signatures of the response to type I and type II interferons (Fig. 5a-c and Extended Data Figs. 9a and 10a; for example, Ifnar2, Irf1, Irf9,Stat1 and Stat2). Type I interferons are critical regulators of B cell homeostasis and responses and potentiate BCR-driven activation, co-stimulation and antigen presentation pathways in B cells . Consistently, we found significant enrichment for BCR signalling (not shown), B cell activation (Lyn, Tnfrsf13c, Btla,
为了确定 B 细胞中 Havcr1 缺失影响 T 细胞介导的抗肿瘤反应的机制，我们分析了 Havcr1 遗传缺失的 B 细胞内在效应。尽管 Havcr 肿瘤、dLN 和 ndLN 中 B 细胞的总频率相对于其各自的对照没有差异（扩展数据图 8a），但来自 dLN 和肿瘤（但不是 ndLN）的 Havcr1 细胞的 scRNA-seq 谱对 I 型和 II 型干扰素的反应特征表达更高（图 5a-c 和扩展数据图 9a 和 10a;例如， Ifnar2、Irf1、Irf9、Stat1 和 Stat2）。I 型干扰素是 B 细胞稳态和反应 的关键调节因子，可增强 B 细胞 中 BCR 驱动的激活、共刺激和抗原呈递途径。一致地，我们发现 BCR 信号传导（未显示）、B 细胞活化 （Lyn、Tnfrsf13c、Btla、

IFN -BUV737 干扰素 -BUV737

b

Percentage of clonally expanded cells among T cells
T 细胞中 克隆扩增细胞的百分比

h

Fig. 4 | Havcr1 deletion in B cells enhances anti-tumour T cell immunity. , Flow cytometry analysis of TILs derived from Cd1 and Havcr1 mice implanted s.c. with B16F10. a, Representative FACS plot and the percentage of IFN and TNF double-positive cells and IL-2 within tumour-infiltrating CD8 (top) and CD4 (bottom) T cells. mice per group.b, Representative FACS plot and the percentage of granzyme and perforin double-expressing T cells. control and mice. , scRNA/BCR-seq and TCR-seq analysis of the TME, dLNs and ndLNs from and mice bearing B16F10 melanoma.c,d,Schematic of the experimental design and UMAP analysis of 11,884 CD cells coloured by their tissue of origin (c) and immune cell types (d).ISG, IFN-stimulated gene; moDCs, monocyte-derived dendritic cells;PMN, polymorphonuclear leukocytes. e, UMAP projection of Cd19 (blue) and (red) cells delineated between conventional cells, cells and cells (left) and clonally expanded cells (middle). Right, the frequencies of clonally expanded CD8 cells in different compartments.f. plot of gene expression comparing versus TILs. Positive -transformed fold change corresponds to upregulation within Havcr1 TILs and vice versa.g, UMAP analysis of TILs coloured by cell types (top left), genotypes (top middle) and clonal expansion (top right). Bottom, expression of the indicated markers. , The frequencies of OVA-specific cells among CD8 TILs (top) and Ki-67-expressing OVA-specific CD8 TILs (bottom). mice per group. Data are mean s.e.m. pooled from at least two to three independent experiments. Statistical analysis was performed using two-tailed Student's -tests and .
图4 |B 细胞中的 Havcr1 缺失可增强抗肿瘤 T 细胞免疫力。 ，流式细胞术分析来自植入 B16F10 皮下注射的 Cd1 和 Havcr1 小鼠的 TIL。a，肿瘤浸润性 CD8 （上）和 CD4 （下）T 细胞中 IFN 和 TNF 双阳性细胞和 IL-2 的代表性 FACS 图和百分比。 每组小鼠.b，代表性FACS图以及颗粒酶 和穿孔素双表达 T细胞的百分比。 对照和 小鼠。 ，来自 携带 B16F10 黑色素瘤的 小鼠的 TME、dLN 和 ndLN 的 scRNA/BCR-seq 和 TCR-seq 分析.c，d，11,884 个 CD 细胞的实验设计和 UMAP 分析示意图，按其来源组织 （c） 和免疫细胞类型 （d） 着色。ISG， IFN刺激基因;moDCs，单核细胞来源的树突状细胞;PMN，多形核白细胞。e，Cd19 （蓝色）和 （红色） 细胞的UMAP投影，在常规 细胞、 细胞和 细胞（左）和克隆扩增 细胞（中）之间 划定。右图为不同区室中克隆扩增的CD8 细胞的频率。 基因表达与 TIL的比较 图。正 转化的倍数变化对应于 Havcr1 TIL 内的上调，反之亦然，按细胞类型（左上）、基因型（中上）和克隆扩增（右上）着色的 TIL 的 UMAP 分析。底部，指示标记的表达。 ，CD8 TILs（上图）和表达Ki-67的OVA特异性CD8 TILs（下图）中OVA特异性细胞的频率。 每组小鼠。数据是平均 s.e.m. 汇集的至少两到三个独立实验。使用双尾 Student's -检验 和 .
Cd81 and Cd22) and antigen processing and T cell antigen presentation and co-stimulation (Icosl, Cd4O and Ciita) gene signatures (Fig. 5a-c and Extended Data Fig. 9b). Supporting these RNA expression patterns, there was increased surface expression of CD86, MHC II and ICOSL on Havcr cells infiltrating the tumours (Extended Data
Cd81 和 Cd22） 和抗原加工以及 T 细胞抗原呈递和共刺激（Icosl、Cd4O 和 Ciita）基因特征（图 5a-c 和扩展数据图 9b）。支持这些 RNA 表达模式的是，浸润肿瘤的 Havcr 细胞上 CD86、MHC II 和 ICOSL 的表面表达增加（扩展数据
Fig. 9c). Although Havcr1 deletion increases the response to type-1 interferons and cell activation, humoral immunity was largely unaffected by its deletion in the tumour setting. Flow cytometry analysis showed similar frequencies of plasmablasts , plasma cells , germinal centre cells or
图9c）。尽管 Havcr1 缺失增加了对 1 型干扰素和 细胞活化的反应，但在肿瘤环境中，体液免疫在很大程度上不受其缺失的影响。流式细胞术分析显示浆母细胞 、浆细胞 、生发中心 细胞 或

C

Up in Havcr Up in Cd19 .
在 Havcr 中 在 CD19 中。

Fig. 5 | TIM-1 deficiency in cells results in cell activation, antigen presentation and co-stimulatory function. a-c, scRNA-seq analysis of B cells derived from TILs, dLNs and ndLNs of and mice bearing B16F10 melanoma. plot of gene expression comparing tumour-derived Cd1 and Haucr1 cells (a), gene set enrichment analysis (GSEA) analysis (b) and dot plots depicting selected genes (c) between tumour-infiltrating Havcr and B cells. Selected genes are annotated. NES, normalized enrichment score. d, peptide-pulsed and B cells were co-cultured with CellTrace Violet (CTV)-labelled OVA-restricted CD4 T cells (OTII) at different ratios for 4 days. T cell proliferation was determined by dilution of CTV. Representative histograms and quantitative analysis of the proliferation indices are shown. mice per group.e, cells were analysed for expression of IFN , ICOS and FOXP3. Representative and quantitative data are shown. The circles denote data points from individual mice. , Naive
图5 |细胞中 TIM-1 缺乏会导致 细胞活化、抗原呈递和共刺激功能。a-c，scRNA-seq 分析来自携带 B16F10 黑色素瘤的 小鼠的 TIL、dLN 和 ndLN 的 B 细胞。 比较肿瘤来源的 Cd1 和 Haucr1 细胞的基因表达图 （a）、基因集富集分析 （GSEA） 分析 （b） 和描述肿瘤浸润性 Havcr 和 B 细胞之间选定基因的点图 （c）。对选定的基因进行注释。NES，归一化富集评分。d、 肽脉冲 细胞和 B 细胞与 CellTrace Violet （CTV） 标记的 OVA 限制性 CD4 T 细胞 （OTII） 以不同比例共培养 4 天。T细胞增殖通过CTV稀释决定。图中显示了具有代表性的直方图和扩散指数的定量分析。 每组小鼠，分析 细胞IFN 、ICOS和FOXP3的表达。显示了代表性和定量数据。圆圈表示来自单个小鼠的数据点。 朴素
CD45.1 OVA-restricted CD4 cells were transferred i.v. 1 day before B16-OVA melanoma cell s.c. implantation into CD45.2 and mice. mice per group. Tumour-infiltrating OTII cells were examined for expression of IFN and FOXP3. A schematic of the experimental and quantitative results is shown. g, Quantification and representative histogram of IFNAR1 surface expression of B cells derived from TILs and dLNs of and Havcr mice implanted s.c. with B16F10. mice per group. , Tumour growth in the indicated mice implanted with B16F10 melanoma and treated with isotype control ( mice per group) or neutralizing anti-IFNAR1 ( mice per group) antibodies. Data are mean s.e.m. pooled or representative of at least two to three independent experiments. Statistical analysis was performed using repeated-measures two-way ANOVA (d and ) and two-tailed Student's -tests .
在 B16-OVA 黑色素瘤细胞皮下植入 CD45.2 和 小鼠前 1 天，将 CD45.1 OVA 限制性 CD4 细胞转移。 每组小鼠。检查肿瘤浸润 OTII 细胞的 IFN 和 FOXP3 表达。图中显示了实验和定量结果的示意图。g，来自植入 B16F10 的 TIL 和 dLN 的 B 细胞以及 Havcr 小鼠的 IFNAR1 表面表达的定量和代表性直方图。 每组小鼠。 ，植入 B16F10 黑色素瘤并用同型对照（每组 小鼠）或中和抗 IFNAR1（每组 小鼠）抗体治疗的指示小鼠的肿瘤生长。数据是平均 s.e.m. 汇总或代表至少两到三个独立实验。使用重复测量双因素方差分析（d 和 ）和双尾学生 检验 进行统计分析。
T follicular helper cells within the and spleen from and control mice (Extended Data Fig. 8b-d,k-m). Furthermore, we did not observe significant differences in circulating immune complexes or in the total amount of or in the serum of either naive or B16F10-bearing Havcr and control mice (Extended Data Fig. 8e-h). Importantly, the levels of B16F10-reactive IgGs and IgM were also unaltered in Havcr sera (Extended Data Fig. 8i). Finally, we did not detect a significant increase in class-switched or clonally expanded B cells across the compartments, and there was no difference in major B cell subsets in Havcr mice or mice treated with anti-TIM-1 monoclonal antibodies (Extended Data Fig. ). Thus, Havcr1 deletion had little to no effect on humoral immunity in tumours and lymphoid organs.
T滤泡辅助细胞 和脾脏内的T滤泡辅助细胞来自 对照小鼠（扩展数据图8b-d，k-m）。此外，我们没有观察到幼稚或携带B16F10的Havcr 和对照小鼠的循环免疫复合物 或血清总量 或 血清中的显着差异（扩展数据图8e-h）。重要的是，Havcr 血清中 B16F10 反应性 IgG 和 IgM 的水平也没有改变（扩展数据图 8i）。最后，我们没有检测到跨区室的类别转换或克隆扩增的B细胞的显着增加，并且在Havcr 小鼠或用抗TIM-1单克隆抗体治疗的小鼠中，主要B细胞亚群没有差异（扩展数据图1）。 ）。因此，Havcr1缺失对肿瘤和淋巴器官的体液免疫几乎没有影响。