医学封面：三阴性乳腺癌杀手猎头

　　三阴性乳腺癌的雌激素受体、孕激素受体、人类表皮生长因子受体HER2均为阴性，对内分泌治疗和HER2靶向治疗无效，药物治疗主要依靠化疗。近年来，免疫细胞程序性死亡受体PD-1及其配体PD-L1（又称免疫检查点）抑制剂通过激活CD8阳性T淋巴细胞（又称细胞毒性或杀手型T淋巴细胞）抗肿瘤活性，对三阴性乳腺癌患者取得良好疗效。不过，免疫检查点抑制剂非常昂贵，而且并非对全部三阴性乳腺癌患者有效。那么，我们如何预测最有可能获益于免疫检查点抑制剂的患者？

　　2023年6月9日，美国细胞出版社旗舰期刊《医学》第4卷第6期以封面文章形式发表复旦大学附属肿瘤医院吴松阳①、张思维①、马丁①、肖毅、刘引、陈力、宋效清、马晓燕、徐颖、柴文君、金希✉、邵志敏✉、江一舟✉等学者的研究报告，首次探讨了树突状细胞对肿瘤免疫微环境的影响及其对三阴性乳腺癌患者免疫治疗结局的影响。该研究利用来自临床试验和小鼠模型的数据，发现以趋化因子CCL19表达为特征的特定功能性树突状细胞状态可以增强CD8阳性T淋巴细胞抗肿瘤活性，并与免疫治疗效果显著相关，肿瘤内和血液循环CCL19水平可以作为预测免疫治疗效果的标志物。本期封面由《分子细胞》学术副主编桑希塔·查克拉博蒂绘制：肿瘤微环境树突状细胞（粉红色）产生CCL19作用于CD8阳性T淋巴细胞（蓝色）可以增强其抗肿瘤效果。

On the cover: In this issue, Wu et al. investigate the role of dendritic cells (DCs) within the tumor immune microenvironment and their impact on immunotherapy outcomes in patients. Using data from clinical trials and mouse models, they identify a specific functional DC state characterized by CCL19 expression that can augment the antitumor activity of CD8+ T cells and is associated with favorable immunotherapy responses. Intratumoral and circulating CCL19 levels may be used as a marker to predict immunotherapy efficacy. On the cover, DCs (in pink) within the tumor microenvironment produce CCL19, which acts on CD8+ T cells (in blue) to enhance their antitumor effectiveness. Cover art: Sonhita Chakrabarty.

　　对此，西班牙应用医学研究中心、纳瓦拉健康研究所、西班牙癌症网络生物医学研究中心发表观点文章：树突状细胞——三阴性乳腺癌抗肿瘤CD8阳性T淋巴细胞的猎头。“猎头”二字形象地反映了树突状细胞对杀手型T淋巴细胞的作用。

　　树突状细胞和抗原交叉启动已被证实对于建立和维持抗癌免疫至关重要，这反过来又在许多临床前环境中促进了免疫检查点抑制剂的有效性。与临床前结果一致，在许多癌症类型中，树突状细胞浸润、CD8阳性T淋巴细胞免疫浸润与良好的整体临床疗效始终相关。复旦研究表明，在三阴性乳腺癌中，显示获得性成熟特征的树突状细胞，与免疫检查点PD-(L)1抑制剂的良好疗效密切相关。重要的是，研究者证明三阴性乳腺癌微环境成熟树突状细胞可产生高水平的趋化因子CCL19。事实上，利用来自三阴性乳腺癌患者免疫检查点抑制剂治疗的若干临床试验数据，研究者证实仅CCL19表达就可确定对PD-(L)1抑制剂疗效良好者。

　　CCL19与其姊妹趋化因子CCL21类似，通过CCR7受体发挥作用，该受体表达于多种免疫细胞群，例如幼稚和中央记忆型T淋巴细胞或成熟树突状细胞，除其他功能外，还介导T淋巴细胞在毛细血管后微静脉中聚集进入次级淋巴器官或淋巴结内T淋巴细胞区的组织。研究者还证实，CCL19阳性树突状细胞与肿瘤内的三级淋巴结构密切相关，表明树突状细胞通过调控免疫微环境空间分布以激活抗肿瘤免疫的潜在作用。这类免疫枢纽（T淋巴细胞、树突状细胞聚集体和三级淋巴结构）存在于肿瘤微环境内或附近以及毛细血管后微静脉（也可以是有组织三级淋巴结构的一部分）已被证明与免疫检查点抑制剂临床疗效良好以及对PD-1抑制剂有效的T淋巴细胞群聚集密切相关。

　　因此，复旦研究结果强调了树突状细胞对肿瘤微环境的重要性：不仅可以启动抗肿瘤T淋巴细胞，还可以聚集最重要的抗肿瘤免疫细胞群，并组织需要特定空间的有效免疫反应，以最大程度引发有效抗肿瘤免疫所需的免疫细胞相互作用。

　　虽然所有相关数据都表明此类组织的关键重要性，但是目前尚不明确允许产生此类免疫细胞枢纽的关键事件是什么，以及肿瘤内这些免疫细胞微环境的形成是否与控制次级淋巴器官形成的机制相同。CCL19阳性树突状细胞浸润于组织与这些免疫细胞枢纽相关，这一事实并未阐明最初需要哪些细胞组织这些组织免疫环境，以及其本身能否指导此类组织形成。该信息对于设计新的治疗方法以最大化肿瘤对免疫检查点抑制剂的敏感性具有重要意义。

　　为此，研究者证明：单独注射CCL19或同时植入表达CCL19的树突状细胞，足以增强至少两种三阴性乳腺癌小鼠模型对免疫检查点抑制剂的敏感性，这表明树突状细胞可能对这些抗肿瘤免疫区域的组织非常重要，或者这些免疫区域通过将树突状细胞聚集于肿瘤微环境发挥其抗肿瘤功能。此处确定的树突状细胞群与已知富含其他肿瘤类型免疫调节分子的成熟树突状细胞高度相似。确定浸润性成熟树突状细胞表达的免疫调节分子是否也可以进行干预以进一步增强其功能，这将是设计治疗干预新措施的重要一步。

　　另一个有待解答的重要问题是：旨在扩大、聚集和促进肿瘤微环境树突状细胞成熟的方法能否使肿瘤适合免疫检查点治疗？扩增树突状细胞的临床前实验（例如FMS样酪氨酸激酶3配体治疗）一直非常有前途，但是此类方法的临床研发仍然处于早期阶段。肿瘤内接种树突状细胞也已经过检测，一些临床前研究试图特异性增强树突状细胞向肿瘤部位聚集。肿瘤微环境是否有足够且完全激活的树突状细胞，足以促进免疫原性较差肿瘤的免疫力，是仍然需要解决的问题。

　　为了设计新的治疗方法，确定肿瘤微环境树突状细胞引发的基本免疫功能非常重要（图）。交叉启动似乎是抗肿瘤CD8阳性T淋巴细胞反应的绝对要求，至少在临床前模型中是这样。复旦研究表明，CCR7阳性T淋巴细胞聚集可能是肿瘤树突状细胞活性的基本机制，并且向肿瘤内注射CCL19本身可以使肿瘤对PD-1抑制剂治疗敏感。之前已经提出树突状细胞（尤其经典1型树突状细胞）对促进免疫浸润的重要性。肿瘤内树突状细胞与T淋巴细胞之间活跃的相互作用已通过活体显微镜和人类癌症标本被详细分析。尽管如此，该相互作用产生的刺激性质尚未完全确定。规避这些功能的策略，例如疫苗接种策略、肿瘤内T淋巴细胞过继疗法或治疗性蛋白质可能是重要的替代方案，尤其因为广泛报道癌症患者缺乏树突状细胞。

肿瘤微环境树突状细胞促进抗肿瘤免疫反应的免疫功能示意图：A、肿瘤内与T淋巴细胞的相互作用，包括抗原呈递和交叉呈递，提供允许激活信号，并维持细胞具有增殖能力。B、抗原摄取并传播至淋巴结以启动和重新激活其他T淋巴细胞。C和D、如复旦研究所示，CCL19等趋化因子产生可吸引特定淋巴细胞群（C）并促进免疫细胞微环境以激活T淋巴细胞（D）

　　复旦研究提供了若干创新发现，对于理解树突状细胞在抗肿瘤免疫中的地位至关重要：首先，确定了树突状细胞对三阴性乳腺癌的重要性；其次，强调树突状细胞对免疫治疗最为重要的肿瘤杀伤细胞——CD8阳性T淋巴细胞聚集的重要性。未来，重要的是要了解树突状细胞相关疗法能否增加免疫治疗获益人群，尤其是目前认为免疫治疗疗效不佳的人群，如肿瘤抗原性较弱的患者。

相关链接

• 一滴血预测三阴性乳腺癌免疫疗效

Med. 2023 Jun 9;4(6):341-343. Impact Score: 20.28

Dendritic cells, headhunters for anti-tumor CD8+ T cells in triple-negative breast cancer.

Carlos Luri-Rey, Almudena Manzanal, Beatrice Pinci, Alvaro Teijeira.

Center for Applied Medical Research (CIMA), Pamplona, Spain; Navarra Institute for Health Research (IDISNA), Pamplona, Spain; Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain.

How do we identify patients most likely to benefit from immune checkpoint blockade therapies? This month in Med, Wu and colleagues identify that CCL19+ mature dendritic cells correlated with responses to anti-PD-(L)1 immunotherapy in triple-negative breast cancer patients, suggesting the use of CCL19 as a biomarker to predict patient outcomes.

Dendritic cells (DCs) and antigen cross-priming have been shown to be essential to set up and sustain anti-cancer immunity, which in turn promotes the effectiveness of checkpoint immune blockade therapy in a number of preclinical settings. In line with preclinical results, DC infiltration consistently correlates with CD8+ T cell immune infiltration and good overall clinical response in many cancer types. Wu and colleagues show that in triple-negative breast cancer (TNBC), the presence of DCs showing features of acquired maturation strongly correlates with a good response to anti-PD-(L)1-based immune checkpoint blockade. Importantly, they demonstrate that mature DCs in the TNBC microenvironment produce high levels of the chemokine CCL19. In fact, using data from several clinical trials of patients with TNBC treated with immune checkpoint therapies, the authors show that CCL19 expression alone can identify good responders to PD-(L)1 blockade. CCL19 (similarly to its sister chemokine CCL21) acts through the CCR7 receptor expressed in several immune cell populations, such as naive and central memory T cells or mature DCs, and mediates, among other functions, the recruitment of T cells across high endothelial venules (HEVs) into secondary lymphoid organs (SLOs) or the organization of the T cell zone within the lymph nodes. The authors were also able to show that the presence of CCL19+ DCs correlated with organized lymphoid aggregates in the tumor microenvironment (TME), including bona fide tertiary lymphoid structures (TLSs), suggesting a potential role for DCs in organizing immune cell microenvironments to sustain anti-tumor immune responses. Both the presence of these types of immune hubs (T cell-DC aggregates and TLSs) within or next to the TME and the presence of HEVs (that can also be part of organized TLSs) have been shown to strongly correlate with good clinical response to immune checkpoint blockade-based therapies and with the recruitment of the T cell populations that are known to respond to PD-1 blockade (Tpex). Therefore, the results of Wu and coworkers emphasize the key importance of DCs in the TME not only to prime anti-tumor T cells but also to recruit the most important anti-tumor immune populations and organize effective immune responses that require a specific spatial organization to maximize the immune cell interactions that are needed to elicit efficient anti-tumor immunity. Despite all the correlative data suggesting the key importance of this type of tissue organization, it is still unclear what the crucial event is that allows the generation of such immune cell hubs and whether the formation of these immune cell microenvironments in the tumors is guided by the same mechanisms that govern SLO formation. The fact that CCL19+ DC infiltration in tissue correlates with these immune cell hubs does not clarify which cells are initially needed to organize these tissue immune environments and whether they themselves can direct such tissue organization. This information will be of great relevance for the design of novel therapeutic approaches to maximize tumor sensitivity to immune checkpoint blockade. In this manuscript, the authors provide proof that CCL19 injection alone or the co-engraftment of DCs expressing CCL19 is sufficient to enhance sensitivity to immune checkpoint blockade in at least two mouse models of TNBC, suggesting that DCs may be very important in the organization of these anti-tumor immune territories or that these immune territories are exerting their anti-tumor functions by recruiting DCs into the TME. The DC population identified here shows a strong similarity with already described mature dendritic cells enriched in immunoregulatory molecules (mregDCs) in other tumor types. Defining whether immunomodulatory molecules expressed by the infiltrating mature DCs could also be amenable to intervention to further potentiate their functions will be an important step toward the design of new therapeutic interventions.

Another important question that remains to be answered is whether approaches aiming to expand, recruit, and promote the maturation of DCs in the TME will render tumors amenable to immune checkpoint therapy. Preclinical experiments pursuing DC expansion, such as Flt3L treatment, have been very promising, but the clinical development of such approaches is still in its early phases. Intratumoral vaccination with DCs has also been assayed, and a few preclinical studies have attempted to specifically enhance recruitment of DCs to the tumor site. Whether having enough and fully activated DCs in the TME is sufficient to promote immunity in poorly immunogenic tumors is a question that still needs to be addressed.

In order to design new therapeutic approaches, it is very important to determine the essential immune functions elicited by DCs in the TME (Figure 1). Cross-priming seems to be an absolute requirement for anti-tumor CD8+ T cell responses, at least in preclinical models. In this study, Wu et al. show that recruitment of CCR7+ T cells is likely a fundamental mechanism behind DC activity in tumors and that intratumoral CCL19 injection itself can render tumors sensitive to anti-PD-1 treatment. The importance of DCs, particularly cDC1s, in promoting immune infiltration has been suggested before. Active interaction of DCs within the tumor with T cells has been well characterized by intravital microscopy and in human cancer specimens. Nonetheless, the nature of the stimuli provided in this dialogue has not yet been completely defined. Strategies to circumvent these functions, such as vaccination strategies, intratumoral delivery of adoptive T cell therapies, or therapeutic proteins, may be important alternatives, especially because of the extensively reported paucity of DCs in cancer patients.

The work of Wu and colleagues provides several important findings that are crucial for understanding the essential role of DCs in cancer immunity: first, by identifying their importance in a less well-explored cancer type, TNBC, and second, by highlighting the importance of DCs in the recruitment of the CD8+ T cells that will be most effective in tumor elimination when treating patients with checkpoint blockade. In the future, it will be important to understand whether therapies promoting DC infiltration and function will expand the number of patients that can benefit from immune checkpoint blockade, particularly if this might include those patients with small chances of benefitting from immunotherapy, such as those with a low antigenic cargo in their tumors.

DOI: 10.1016/j.medj.2023.05.004