Live or Let Die: T Cell Survival in Cancer Immunotherapy

活着還是死去：生存在癌症免疫療法中的T細胞

作者 | Janne Ruotsalainen and Thomas Tuting

編譯 | Aaron

來源 | Immunity 50, February 19, 2019（IF=19.7）

https://doi.org/10.1016/j.immuni.2019.01.016

Combination immune checkpoint blockade targeting CTLA-4 and PD-1 is thought to reinvigorate exhausted T cells better than monotherapies. In this issue of Immunity, Pai et al. (2019) show that, in the setting of low tumor burden, this combination regimen promotes interferon-γ-dependent T cell hyperactivation and death and thus favors tumor progression. 

靶向CTLA-1和PD-1的組合免疫檢查點阻斷被認爲比單一療法更好地重建耗竭的T細胞。在本期《Immunity》中，Pai等研究表明，在低腫瘤負荷環境下，該組合方案促進幹擾素-γ-依賴性T細胞過度活化和死亡，從而有利於腫瘤惡化。

Immune checkpoint blockade (ICB) targeting the immunoregulatory molecules CTLA-4 and PD-1 on T cells is transforming cancer treatment. However, durable treatment responses are only observed in a subset of patients. Understanding the mechanisms of therapy resistance is a key challenge in the field. T-cell-inflamed, ‘‘hot’’ tumors with local induction of interferon (IFN)-stimulated genes, including PD-L1 and a high number of neoantigens, are the most likely to respond to ICB therapy. The magnitude of T cell reinvigoration in relation to pretreatment tumor burden correlates with clinical responses to ICB. In this issue of Immunity, Pai et al. (2019) report the surprising observation that combination ICB targeting both CTLA-4 and PD-1 is less effective than ICB targeting CTLA-4 alone in mice with low tumor burden (LTB). Mechanistically, they discovered that increased amounts of therapy-induced IFN-γ production can promote deletion of dominant tumorspecific T cells by enhancing activationinduced cell death (AICD). These findings reveal a mechanism of ICB resistance related to tumor burden, which has important clinical implications.

靶向T細胞上免疫調節分子CTLA-4和PD-1的免疫檢查點阻斷（ICB）正在改變癌症治療。然而，僅僅在一組病人中觀察到持久的治療響應。理解治療抗性的機制是該領域的關鍵挑戰。受到局部誘導幹擾素（IFN）刺激的基因（包括PD-L1和大量新抗原）的T細胞發炎，“熱”腫瘤最有可能對ICB治療有響應。關於預處理腫瘤負荷的T細胞重建程度關係到ICB的臨牀響應。在本期《Immunity》，Pai等報道了令人驚訝的觀察結果，即在低腫瘤負荷（LTB）小鼠中同時靶向CTLA-4和PD-1的組合ICB效果低於僅針對CTLA-4的ICB。從機制上來說，他們發現增加治療誘導產生的IFN-γ的量能夠通過增強激活誘導細胞死亡（AICD），促進顯性腫瘤特異性T細胞的缺失。這些發現揭示了與腫瘤負荷相關ICB抗性的機理，具有重要的臨牀意義。

In their investigations, Pai et al. (2019) utilized TRAMP-C2 prostate cancer cells derived from genetically engineered mice as well as MOC-1 cancer cells derived from a carcinogen-induced mouse squamous cell carcinoma. When transplanted in the skin of syngeneic mice, both cancer cell lines generate T-cell-inflamed tumors in vivo. Mice with high tumor burden (HTB) showed better responses to anti-PD-1 and anti-CTLA-4 combination immunotherapy than did mice given the respective monotherapies. However, in mice with low tumor burden (LTB), combination ICB therapy was inferior to anti-CTLA-4 monotherapy. A retrospective analysis of data from 153 advanced melanoma patients confirmed that anti-PD-1 and anti-CTLA-4 combination immunotherapy was less effective in patients with LTB than in patients with HTB. In contrast, anti-PD-1 monotherapy was effective in both patient subsets. Collectively, these results suggest a potentially detrimental effect of combination ICB in early tumor stages.

在Pai等的研究中，利用了源自基因工程小鼠的TRAMP-C2前列腺癌細胞以及源自致癌物誘導的小鼠鱗狀細胞癌的MOC-1癌細胞。當移植到同系小鼠皮膚中時，這兩種癌細胞株都在體內產生T細胞炎症性腫瘤。高腫瘤負荷（HTB）的小鼠顯示了對抗PD-1和抗CTLA-4組合免疫療法比分別接受單一療法的小鼠更好的響應。然而，在低腫瘤負荷（LTB）小鼠中，組合ICB療法不如抗CTLA-4單一療法。對153例晚期黑色素瘤患者的回顧性數據分析證實，抗PD-1和抗CTLA-4組合免疫療效在低腫瘤負荷患者低於高腫瘤負荷患者。相反，抗PD-1單一療法在上述兩個患者亞組中均有效。總體看來，這些結果表明組合ICB在早期腫瘤階段的可能有潛在不利影響。

In subsequent experiments, the authors investigated the expansion of CD8+ T cells recognizing the immunodominant tumor-rejection antigen SPAS-1 and the subdominant tumor antigen SPAS-2. Mice treated with anti-CTLA-4 monotherapy in the LTB setting showed persistent induction of SPAS-1-specific CD8+ T cells. However, mice treated with combination ICB only showed transient induction of SPAS-1-specific CD8+ T cells that were lost over time. T cell responses directed against SPAS-2 could be persistently induced in all treatment groups. Analyses of T cell receptor (TCR) diversity confirmed a shift from high-abundance T cell clones toward low-frequency clones in mice treated with combination ICB in the LTB setting. Detailed investigations in an LTB metastatic melanoma patient who received combination ICB revealed a similar kinetic of tumor-antigen-specific T cells, which paralleled the clinical course, providing further clinical evidence for a Immunity potential detrimental effect of combination ICB in early tumor stages.

在隨後實驗中，作者研究了識別免疫顯性腫瘤排斥抗原SPAS-1和亞顯性腫瘤抗原SPAS-2的CD8+T細胞擴增。在LTB環境中用抗-CATL-4單一療法治療小鼠顯示對SPAS-1特異性CD8+T細胞持續誘導作用。然而，用組合ICB處理的小鼠僅顯示了對SPAS-1特異性CD8+T細胞的瞬時感應，隨着時間推移而丟失。所有治療組均存在可持續誘導針對SPAS-2的T細胞反應。T細胞受體（TCR）多樣性分析證實了在LTB環境下，ICB組合治療小鼠從高丰度T細胞克隆向低頻克隆的轉變。對一位接受了組合ICB治療的LTB轉移性黑色素瘤患者的詳細研究顯示，腫瘤抗原特異性T細胞具有與臨牀過程類似的動力學，爲組合ICB在早期腫瘤階段的免疫潛在不利作用提供了進一步的臨牀證據。

To gain further insights into the underlying mechanisms, Pai et al. (2019) examined transcriptional profiles of SPAS-1-specific CD8+ T cells isolated 28 days after tumor inoculation from draining lymph nodes of mice receiving ICB treatments. Analyses of differentially expressed genes revealed activation of multiple pathways associated with T cell apoptosis and cell death in combination ICB cohorts when compared with monotherapy cohorts. Analyses of serum cytokine levels showed higher amounts of IFN-γ in mice receiving combination ICB than in those receiving monotherapy. IFN-γ treatment was subsequently shown to promote cell death in mature antigen-specific T cells in vitro, suggesting that IFN-γ contributed to the deletion of tumor-antigenspecific T cells.

爲了進一步瞭解潛在的機制，Pai等檢測了從接受ICB治療小鼠引流淋巴結腫瘤接種28天后分離的SPAS-1特異性CD8+ T細胞的轉錄譜。差異表達基因的分析顯示ICB組合組與單一治療組相比對T細胞凋亡和細胞死亡相關的多通路激活。對血清細胞因子水平的分析表明接受ICB組合治療的小鼠中比接受單一治療的小鼠有更高數量的IFN-γ。隨後，IFN-γ治療被證明可以促進成熟的抗原特異性T細胞體外死亡，這表明IFN-γ有助於腫瘤抗原特異性T細胞的缺失。

Subsequently, Pai et al. (2019) explored the role of IFN-γ signaling for the efficacy of combination ICB in the LTB setting by using mice lacking IFN-γ-receptors (Ifngr1/ mice). The therapeutic efficacy was significantly greater in Ifngr1/ mice than in wild-type mice. These data seem at first counterintuitive, considering that IFN-γ is the prototypic Th1 effector cytokine and that antibody-mediated neutralization of IFN-γ has been consistently shown to suppress immunotherapy efficacy. However, it is becoming increasingly clear that unlike type I IFNs, which are required for activation of innate and adaptive immune cells in the host, IFN-γ mediates its effects directly on tumor cells by suppressing cell proliferation and stimulating antigen presentation. Using bone marrow chimeras, the authors subsequently showed that the increased efficacy of combination ICB in the LTB setting depended on intact IFN-γ receptor signaling in immune cells. Combination ICB treatment of wild-type (CD45.1-congenic) mice reconstituted with Ifngr1/ (CD45.2) bone marrow cells in the LTB setting revealed SPAS-1-specific (but not SPAS-2-specific) CD8+ T cells to be predominantly derived from Ifngr1/ cells, indicating their preferential expansion upon combination therapy. These experimental results support the hypothesis that upon combination ICB in the LTB setting, CD8+ T cells specific to strong tumor antigens can experience deletion through IFN-γ-mediated hyperactivation and AICD.

隨後，Pai等使用缺乏IFN-γ的小鼠(ifngr1/小鼠)，研究了IFN-γ信號對組合ICB治療在LTB環境中的功效作用。對ifngr1/小鼠的治療功效明顯大於野生小鼠。考慮到IFN-γ是Th1效應細胞因子的原型，以及IFN-γ的抗體介導中和一直顯示抑制免疫治療效果，這些數據乍一看與直覺相悖。然而，越來越清晰的是，不同於需要激活宿主先天和適應性免疫細胞的I型幹擾素，IFN-γ通過抑制細胞增殖和刺激抗原遞呈直接介導其對腫瘤細胞的作用。通過使用骨髓嵌合體，作者隨後表明ICB組合治療在LTB環境下功效的提升取決於免疫細胞中完整的IFN-γ受體信號。在LTB環境下對用Ifngr1/ (CD45.2)骨髓細胞重組的野生小鼠（CD45.1-同系）的ICB組合治療顯示SPAS -1特異性(但不是SPAS -2特異性) CD8+ T細胞主要來源於Ifngr1/細胞，表明其在組合治療時優先擴增。這些實驗結果支持瞭如下假設，在LTB環境下ICB組合治療時，特定於強腫瘤抗原的 CD8+ T 細胞可以通過IFN-γ介導的過度激活和AICD發生缺失。

Mechanisms of resistance to immunotherapy largely overlap the mechanisms whereby tumors evade immune recognition during the natural course of disease. These resistance mechanisms include tumor-cell-intrinsic alterations such as loss of tumor antigen presentation, insensitivity to IFNs, dedifferentiation, and increased resistance to apoptosis, as well as tumor-cellextrinsic, microenvironmental alterations such as recruitment of regulatory T cells and myeloid immunosuppressive cells associated with increased expression of immunosuppressive cytokines and metabolites. Therapy-induced deletion of tumor-antigen specific T cells as described in the work by Pai et al. (2019) represents an additional, immune-cell-intrinsic mechanism that could be particularly important after early therapeutic intervention. Berner et al. (2007) reported a similar IFN-γ-dependent apoptosis of CD4+ T cells in mice immunized with tumor cells and treated with agonistic CD40 antibody and IL-2. This immunotherapy regimen initially proved to be protective, but mice were later unable to mount effective vaccine-induced memory responses. This loss of tumor-antigen-specific immunity could well be explained by IFN-γ-dependent T cell hyperactivation and death.

免疫療法的抗性機制很大程度上與在疾病自然過程中的腫瘤躲避免疫識別憑藉的機制重疊。這些抗性機制包括腫瘤細胞內部的改變，如腫瘤抗原表達的缺失，幹擾素不敏感，去分化和增強細胞凋亡抗性，以及腫瘤細胞外在微環境的改變，如調節性T細胞和增加免疫抑制細胞因子及代謝物表達的骨髓免疫抑制細胞的補充。如Pai等工作中所述，治療誘導的腫瘤抗原特異性T細胞缺失代表另外一種免疫細胞內在機理，可能在早期幹預治療後尤爲重要。Berner等報道了在使用腫瘤細胞免疫和用激動劑CD40抗體和IL-2處理後的小鼠CD4+T細胞中，發現了一種類似的IFN-γ依賴性細胞死亡。這種免疫治療方案起初證明是有保護作用的，後來，小鼠無法進行有效的疫苗誘導記憶反應。這一腫瘤抗原免疫性缺失可以用IFN-γ依賴性T細胞過度激活和死亡來很好的解釋。

Induced death of effector T cells is considered to be a physiologically relevant mechanism that helps to maintain self-tolerance by promoting the deletion of potentially autoreactive T cells. This hypothesis was initially proposed by Liu and Janeway (1990), who provided the first experimental evidence that IFN-γ might play an important role in this process. They speculated that the requirement for IFN-γ indicates that this self-tolerance mechanism most likely applies to activated rather than naive T cells and thereby complements clonal deletion of developing T cells in the thymus and clonal anergy of naive T cells as mechanisms to eliminate autoreactivity. Fas-FasL interactions are crucially important for AICD of T cells, and it is conceivable that the requirement for IFN-γ is indirect. However, similar to the findings by Pai et al. (2019), IFN-γ promotes the deletion of antigen-specific CD8+ T cells and limits the size of the memory cell population in an experimental mouse model for acute influenza virus infection (Prabhu et al., 2013). This demonstrates that IFN-γ also has an important role in T cell contraction.

誘導效應T細胞死亡被認爲是一種生理相關的機制，通過提升潛在自反應性T細胞的缺失來保持自我耐受性。這一假設最初由Liu和Janeway提出，並提供了IFN-γ可能在這一過程中發揮重要作用的第一個實驗證據。他們推測對IFN-γ的要求表明這一自我耐受性機理最可能應用於活化T細胞而不是初始T細胞，從而補充胸腺中發展 T 細胞的克隆缺失和初始T細胞的克隆失效作爲消除自反應的機制。Fas-FasL相互作用的T細胞的AICD有着至關重要的作用，可以想象對IFN-γ的需要是間接的。然而，與Pai等人的發現類似，在急性流感病毒感染的實驗小鼠模型中，IFN-γ提促進了抗原特異性CD8+T細胞缺失，並限制了記憶細胞的尺寸。這說明瞭IFN-γ在T細胞收縮方面也有重要作用。

The observation that IFN-γ can limit cellular anti-viral and anti-tumor immunity is unexpected. This cytokine is mainly produced by activated T cells as well as natural killer (NK) cells and plays a key role in eliminating virally infected or transformed cells. One explanation for the differential effects on cellular immunity is the timing of IFN-γ-mediated responses in peripheral tissues. In initial disease stages, IFN-γ is critical for the induction of CXCR3-binding chemokines and thereby contributes to the recruitment of cytotoxic T cells and NK cells. IFN-γ also promotes macrophage cytotoxicity and restricts blood supply. However, in progressive disease, IFN-γ promotes counter-regulatory mechanisms that are thought to limit tissue destruction. An example is the recruitment of T-cell suppressive neutrophils that upregulate the inhibitory receptor PD-L1 as well as T-cell-suppressive metabolic enzymes such as IDO and INOS (Glodde et al., 2017). Collectively, this initiates a regenerative response that attempts to restore tissue homeostasis, a process resembling physiologic wound healing. On the basis of this model, it is tempting to speculate that the nature and magnitude of immunosuppressive networks, which are established over time in progressively growing tumors, critically determines the optimal amount of IFN-γ induction needed for effective ICB therapies. Administration of combination ICB shortly after inoculation of immunogenic TRAMP tumor cells in mice, as performed by Pai et al. (2019) to experimentally model the LTB setting, might well result in strong IFN-γ responses that compromise the survival of antigen-specific CD8+ effector T cells. Future studies will have to clarify the stage of T cell activation and the nature of the microenvironment where T cells are most vulnerable for IFN-γ-induced AICD following combination ICB. These studies also need to carefully exclude the confounding effects of local inflammatory responses, which occur during the first days after tumor cell inoculation.

IFN-γ能抑制細胞抗病毒和抗腫瘤免疫的研究結果令人意外。這種細胞因子主要由活化T細胞和自然殺傷細胞產生，並在消除病毒感染或轉化的細胞有重要作用。細胞免疫不同功效的一種解釋是外圍組織中IFN-γ介導響應的時間。在疾病初期，IFN-γ是誘導CXCR3結合趨化因子的關鍵，因此有助於細胞毒性T細胞和NK細胞的補充。IFN-γ還促進巨噬細胞的細胞毒性並限制血液供應。然而，在疾病發展過程中，IFN-γ促進被認爲是限制組織破壞的反調節機制。一個實例是T細胞抑制中性粒細胞的補充，上調抑制受體PD-L1，以及T細胞抑制代謝酶，如IDO和INOS。總而言之，這引發了再生響應，試圖恢復組織穩態，類似於生理性傷口癒合的過程。在此模型的基礎上，容易推測在逐漸增長的腫瘤中隨着時間建立的免疫抑制網絡的性質和規模，其關鍵是確定有效的ICB治療所需的IFN-γ誘導的最佳量。對接種免疫原性TRAMP腫瘤細胞後不久的小鼠進行ICB組合用藥，如同Pai等實驗的那樣，在實驗上模擬LTB環境，很可能導致強烈的IFN-γ響應，即破壞抗原特異性CD8+效應T細胞的存活。未來的研究將要明確T細胞激活的階段和微環境的本質，其中在組合ICB後，T細胞在組合ICB後最易受IFN-γ誘導的AICD的影響。這些研究也需要謹慎地排除局部炎症反應的混雜效應，這種效應發生在腫瘤細胞接種後的第一天。

The work of Pai et al. (2019) demonstrates that combination ICB cancer immunotherapy can potentially be detrimental in early disease stages with LTB due to increased IFN-γ production, which Immunity leads to T cell hyperactivation and subsequent deletion of dominant tumor-specific T cells (Figure 1). These findings are particularly relevant for ongoing clinical trials that examine the efficacy of ICB therapies in the neoadjuvant setting (Robert, 2018). Priming of naive and memory T cells with cancer vaccines targeting neoantigens could also be particularly vulnerable for IFN-γ-dependent clonal deletion of high-affinity T cells after combination ICB. Recently, novel experimental techniques have been developed to allow us to understand T cell dysfunction in cancer tissues at the singlecell level and to follow the dynamic evolution of clonal T cell responses (Thommen and Schumacher, 2018). 

Pai等的工作說明瞭在LTB環境下，ICB組合癌症免疫療法在疾病早期可能是潛在有害的，因爲IFN-γ產生增加，其免疫作用導致的T細胞過度激活和隨後的顯性腫瘤特異性T細胞缺失（圖1）。這些發現特別與正在進行的臨牀實驗相關，以檢查在新輔助治療中ICB治療的療效。以靶向新抗原的癌症疫苗引發初始T細胞和記憶T細胞，也可能特別容易受到組合ICB治療後高親和力T細胞的IFN-γ依賴克隆缺失的影響。最近，已經開發了新型實驗技術讓我們在單細胞層面理解癌症組織中的T細胞功能紊亂，以及跟蹤克隆T細胞響應的動態演化。

These approaches will undoubtedly shed new light on the relative importance of different mechanisms that mediate immunotherapy resistance and help to design personalized strategies for achieving optimal stimulation of antitumor immunity.

這些方法將毫無疑問對介導免疫治療抗性不同機制的相對重要性做出新的闡述，並幫助設計個性化策略，以實現最佳的抗腫瘤免疫刺激。

If you are interested in pharmaceutical industry, if you've already known the industry well, welcome to join us. 如果您立志在醫藥行業發展, 如果您已在此行業身經百戰, 歡迎加入我們的行列. We are Tony, Rona, Aaron, Vivian, Effy, Brynn...(Your Name)