NK cell-triggered CCL5/IFN?-CXCL9/10 axis underlies the clinical efficacy of HER2-targeted antibodies in primary HER2 positive breast cancer [RNA-seq]

Tumor-infiltrating (TI)-NK cell numbers predict better than TIL score the efficacy of HER2-targeted antibodies in primary breast cancer patients. To understand the mechanism/s underlying this association, biological processes enriched in NK cell-infiltrated as compared to NK cell-desert HER2-positive breast tumors paired by TIL score were leveraged from transcriptomic data. NK cell-infiltrated tumors were enriched in transcripts regulated by interferons and NF-kB. Among them, levels of CCL5/IFNG-CXCL9/10 positively correlated with the number of TI-NK cells in the original biopsy. Indeed, coordinated expression of CCL5/IFNG-CXCL9/10 transcripts was also evidenced in tumor biopsies from a phase II clinical trial where IFNG levels associated to the achievement of pathological complete response to anti-HER2 antibody treatment (OR 96.3, p=0.01) and correlated with their TI-NK cell score. In in vitro models, anti-HER2 antibody-dependent NK cell activation led to the secretion of CCL5/IFN-? and the subsequent production of IFN-?-dependent CXCL9/10 by bystander breast cancer cells. Ex vivo treatment of breast tumor-derived multicellular cultures with trastuzumab induced the activation of CD16+ TI-NK cells and their conversion into a CD16-CD103+ subpopulation, both endowed with CCL5 and IFN-? producing potential. CD16+ and CD16-CD103+ TI-NK cell subpopulations shared the expression of EOMES, TBX21 and several KIR genes, indicating their lineage relationship; and their proportions positively correlated with total NK cell, CD8+ and tissue-resident T cell frequencies, immune cell subsets with anti-tumor potential. Remarkably, the coordinated induction of CCL5/IFNG-CXCL9/CXCL10 expression, concomitant to the conversion of CD16+ into CD16+/-CD103+ tumor-infiltrating NK cells, was recapitulated in a humanized HER2+ breast cancer in vivo model treated with a combination of anti-HER2 antibodies and in vitro expanded human NK cells, paralleling tumor growth control. Finally, patients achieving good clinical responses to anti-HER2 antibody-based neoadjuvant treatment showed an early and coordinated increase in sera CCL5 and CXCL9/CXCL10 levels which positively correlated with TI-NK cell numbers in the corresponding diagnosis biopsy. Overall, our data point to NK cells as regulators of the tumor microenvironment through the early secretion of CCL5/IFN-? resulting in the production of CXCL9/10 and the recruitment/differentiation of immune infiltrates with anti-tumor potential, ultimately contributing to anti-HER2 antibody clinical efficacy. Overall design: Paired samples of peripheral blood and treatment-naive breast tumor specimens were obtained from 3 breast cancer patients, blind to their clinicopathological features. Multicellular suspensions from tumors were obtained as described above and stained with directly labelled antibody cocktail including a-CD45-AF700 (clone 2D1), a-CD3-PerCP (clone SK7), a-CD56-APC (clone CMSSB), a-CD16-efluor780 (clone CB16), a-CD103-FITC (Clone B-Ly7) and DAPI as a viability die. Distinct tumor-infiltrating NK cell subsets were sorted based on the expression of CD16 and CD103 (CD16+; CD16-CD103+; CD16-CD103-) from the CD56+ CD3 gate in CD45+ DAPI- lymphocytes. Peripheral blood NK cells were sorted from PBMC based on their CD56bright CD16- and CD56dim CD16+ expression profile. Cell sorting was performed in a Influx sorter ( BD, YYY) at the Flow Cytometry Facility, PRBB, Barcelona.

肿瘤浸润性（Tumor-infiltrating, TI）NK细胞数量相较于肿瘤浸润淋巴细胞（Tumor Infiltrating Lymphocyte, TIL）评分，更能准确预测原发性乳腺癌患者接受抗人表皮生长因子受体2（HER2）抗体治疗的临床疗效。为阐明该关联背后的潜在机制，本研究借助转录组学数据，对比了匹配TIL评分的NK细胞浸润型与NK细胞缺失型HER2阳性乳腺肿瘤，筛选出在NK细胞浸润型肿瘤中富集的生物学过程。NK细胞浸润型肿瘤中，受干扰素与NF-κB调控的转录本显著富集。其中，CCL5/IFNG-CXCL9/10的表达水平与原始活检样本中的TI-NK细胞数量呈正相关。研究证实，在一项II期临床试验的肿瘤活检样本中，同样观察到CCL5/IFNG-CXCL9/10转录本的协同表达；其中IFNG水平与抗HER2抗体治疗后获得病理完全缓解（pathological complete response, pCR）显著相关（优势比OR=96.3，P=0.01），且与患者的TI-NK细胞评分呈正相关。

在体外模型中，抗HER2抗体依赖的NK细胞活化可促使NK细胞分泌CCL5与IFN-γ，旁观者乳腺癌细胞随后会产生IFN-γ依赖的CXCL9/10。对乳腺肿瘤来源的多细胞培养物施以曲妥珠单抗（trastuzumab）离体处理，可诱导CD16+ TI-NK细胞活化，并将其转化为CD16-CD103+亚群，两类亚群均具备分泌CCL5与IFN-γ的能力。CD16+与CD16-CD103+ TI-NK细胞亚群共同表达EOMES、TBX21及多种杀伤细胞免疫球蛋白样受体（Killer cell Immunoglobulin-like Receptor, KIR）基因，提示二者具有谱系相关性；且二者的比例与总NK细胞、CD8+ T细胞及组织驻留T细胞的频率呈正相关，而这些免疫细胞亚群均具备抗肿瘤活性。

值得注意的是，在联合使用抗HER2抗体与体外扩增人NK细胞治疗的人源化HER2+乳腺癌体内模型中，同样重现了CCL5/IFNG-CXCL9/CXCL10的协同诱导表达，以及CD16+ TI-NK细胞向CD16+/-CD103+肿瘤浸润性NK细胞的转化，该现象与肿瘤生长控制效果相一致。最终，接受基于抗HER2抗体的新辅助治疗（neoadjuvant treatment）并获得良好临床应答的患者，其血清中CCL5与CXCL9/CXCL10的水平会出现早期且协同的升高，且该升高水平与对应诊断活检样本中的TI-NK细胞数量呈正相关。

综上，本研究数据表明，NK细胞可通过早期分泌CCL5与IFN-γ调控肿瘤微环境，进而诱导CXCL9/10的产生，并招募/分化出具备抗肿瘤活性的免疫浸润细胞，最终助力抗HER2抗体的临床疗效。

实验设计：本研究从3名乳腺癌患者中获取了外周血与未经治疗的乳腺肿瘤标本的配对样本，所有样本均未知晓其临床病理特征。按照前述方法制备肿瘤多细胞悬液，使用直接标记的抗体组合进行染色，包括抗CD45-AF700（克隆号2D1）、抗CD3-PerCP（克隆号SK7）、抗CD56-APC（克隆号CMSSB）、抗CD16-efluor780（克隆号CB16）、抗CD103-FITC（克隆号B-Ly7），以及作为死活染料的DAPI。在CD45+ DAPI-淋巴细胞的CD56+ CD3-门中，根据CD16与CD103的表达情况，分选得到不同的肿瘤浸润性NK细胞亚群（CD16+；CD16-CD103+；CD16-CD103-）。从外周血单个核细胞（Peripheral Blood Mononuclear Cell, PBMC）中，根据CD56bright CD16-与CD56dim CD16+的表达谱，分选得到外周血NK细胞。细胞分选在巴塞罗那PRBB流式细胞术设施的Influx分选仪（BD公司）中完成。