个体化新抗原特异性T细胞过继免疫：任重道远，砥砺前行

近年来,肿瘤新抗原掀开了个体化免疫治疗的新篇章,作为基于新抗原个体化免疫治疗的重要组成部分,新抗原特异 性T细胞的过继输注（ACT）疗法备受瞩目。本文将首先从新抗原特异性T细胞ACT治疗应用策略及临床应用现状介绍新抗原 特异性T细胞ACT治疗这一新兴的精准免疫治疗的发展现状,然后从新抗原的预测、新抗原特异性T细胞筛选及扩增等方面系 统地总结新抗原T细胞ACT治疗所面临的阻碍和挑战,最后从优化新抗原预测、增加新抗原特异性T细胞数量和多样性、防止新 抗原特异性T细胞过度分化或死亡、缩短生产周期和减少生产成本及探索联合治疗方式等五个方面对该领域的未来发展机遇和 研究方向进行重点阐述。     

Mechanisms and strategies for safe chimeric antigen receptor T-cell activity control

The clinical application of chimeric antigen receptor (CAR) T-cell therapy has rapidly changed the treatment options for terminally ill patients with defined blood-borne cancer types. However, CAR T-cell therapy can lead to severe therapy-associated toxicities including CAR-related hematotoxicity, ON-target OFF-tumor toxicity, cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). Just as CAR T-cell therapy has evolved regarding receptor design, gene transfer systems and production protocols, the management of side effects has also improved. However, because of measures taken to abrogate adverse events, CAR T-cell viability and persistence might be impaired before complete remission can be achieved. This has fueled efforts for the development of extrinsic and intrinsic strategies for better control of CAR T-cell activity. These approaches can mediate a reversible resting state or irreversible T-cell elimination, depending on the route chosen. Control can be passive or active. By combination of CAR T-cells with T-cell inhibiting compounds, pharmacologic control, mostly independent of the CAR construct design used, can be achieved. Other strategies involve the genetic modification of T-cells or further development of the CAR construct by integration of molecular ON/OFF switches such as suicide genes. Alternatively, CAR T-cell activity can be regulated intracellularly through a self-regulation function or extracellularly through titration of a CAR adaptor or of a priming small molecule. In this work, we review the current strategies and mechanisms to control activity of CAR T-cells reversibly or irreversibly for preventing and for managing therapy-associated toxicities.     

Antitumor effector functions of T cells are dependent on in vivo priming and restricted T-cell receptor expression

Tumor-specific T cells are crucial for immunologic control of malignant disease. T cells can be induced in vivo by vaccination or adoptively transferred after activation ex vivo. We investigated the requirements for generating T cells with optimal antitumor effector functions in a murine lymphoma model. Using adoptive transfer, we show that in vivo efficacy of T cells cannot be predicted by tumor reactivity in vitro. A restricted T-cell receptor beta chain repertoire of T-cell populations stimulated ex vivo against tumor cells was necessary but not sufficient for tumor protectivity. Tumor elimination furthermore required vaccination of donor mice, hence in vivo priming. The in vivo priming step may allow tumor-specific T cells to accumulate in vitro more rapidly and to survive for longer periods after withdrawal of the antigenic stimulus and adoptive transfer. A possible survival benefit of in vivo induced T cells may be ascribed to the responsiveness to homeostatic cytokines and to unique cytokine milieus encountered in vivo. Most importantly, monoclonal T cells cannot inhibit tumor growth. A prerequisite of tumor rejection was the expression of at least 2 T-cell receptor beta chains by transferred T-cell populations. This finding has implications for designing adoptive transfer strategies for the clinic.     

NK细胞肿瘤免疫治疗技术的挑战

自然杀伤(NK)细胞具有以MHC I 非依赖识别机制和快速杀伤病变细胞能力、低移植物抗宿主反应(GVHD)风险、可采用异体细胞回输、体内存活周期短和无细胞因子风暴等长期和不可预期风险较低等特点和优势,使其在肿瘤免疫治疗中展现出巨大的应用潜力。虽然外周血单个核细胞（PBMC）来源NK细胞相比干细胞来源NK和NK细胞系在安全性和肿瘤杀伤能力上相对更好,但细胞制备技术的效率、稳定性和安全性仍有待完善;NK细胞被认为是较理想的嵌合抗原受体（chimeric antigen receptor,CAR）载体,但外周血来源NK细胞转染效率较低,影响了CAR-NK的研发进程。由于NK细胞来源和培养技术的多样性,使细胞制品的活性不一,虽然NK细胞在抗血液肿瘤治疗中表现相对突出,但对实体瘤的治疗效果仍有待验证。总之,NK细胞应用开发近年已取得显著进步,但仍面临生产技术和临床疗效等诸多挑战。     

化疗联合细胞因子诱导的杀伤细胞治疗中晚期胃癌临床研究

目的评估化疗联合细胞因子诱导的杀伤细胞（cytokine induced killers，CIK）治疗中晚期胃癌的临床疗效。方法参照美国斯坦福大学骨髓移植中心建立的CIK培养方法诱导扩增CIK联合化疗，培养14d后分次回输给病人，观察病人治疗前后瘤体变化、生活质量、卡氏评分等，同时记录生存期。结果21例胃癌单纯化疗后，完全缓解（CR）2例，部分缓解（PR）4例，好转（MR）8例，稳定（SD）3例，无改变（PD）4例，总有效率为28．6％。21例化疗联合CIK治疗后，完全缓解（CR）4例，部分缓解（PR）9例，好转（MR）6例，稳定（SD）1例，无改变（PD）1例，总有效率为52．38％。两组之间具有明显的差异（x^2=6．43，P〈0．05），胃癌单纯化疗后1a生存期为90．5％，2a生存期为81．0％，3a生存期为71．4％。胃癌化疗联合CIK治疗后1a生存期为95：2％，2a生存期为85．7％，3a生存期为81．0％。两组3a生存期没有明显差异（P〉0．05）。用Karnofsky评分及体重的改变来评估生存质量的改善。胃癌单纯化疗后Karnofsky评分平均提高10分，其中提高5例，稳定8例，下降8例，提高率为23．8％。化疗联合CIK治疗后Karnofsky评分平均提高10-20分，其中提高10例，稳定5例，下降6例，提高率为47．6％。两组生存质量变化没有明显差异（P〉0．05）。结论化疗联合CIK治疗对为那些手术、放疗、单纯化疗已无适应证的病人提供了一种可以继续延长生存期，提高生活质量的新的途径。     

Locoregional immunotherapy in cancer patients: Review of clinical studies

Many patients with invasive cancer have a compromised immune system. Thisimmune dysfunction does appear to start at the site of the tumor. Locoregionalimmunotherapy is given to stimulate the immune system in order to kill tumorcells either indirectly via a specific or a non-specific way or directly viacell transfer therapy. Advantages to give this immunotherapy locoregionallyin stead of systemically are a higher concentration of the immunomodulator atthe site of the tumor, to attract or activate effector cells, and diminishedtoxicity. In this review we have summarised the clinical studies usingloco-regional immunotherapy in patients with cancer. Only phase I and IIstudies were performed. Clinical responses were seen. No single locoregionaltreatment has become a standard therapy. Relatively few investigations wereperformed to estimate the influence on the locally effector mechanisms orimmune dysfunction. In future clinical trials it is essential to get a betterinsight in these mechanisms.     

B-cell maturation antigen-specific chimeric antigen receptor T cells for multiple myeloma: Clinical experience and future perspectives

Despite major advances in the treatment of multiple myeloma (MM), it remains a largely incurable disease with long-term control often dependent on continuous therapy. More effective, better tolerated treatments are therefore required to achieve durable remissions and to improve the quality of life of MM patients. Adoptive immunotherapy employing T cells expressing chimeric antigen receptors (CAR) is currently among the most promising treatment approaches in cancer. Within the target portfolio for MM immunotherapy, B-cell maturation antigen (BCMA) is among the most widely studied target antigens. BCMA is consistently expressed on MM cells and, importantly, is not expressed in critical healthy tissue. For this reason, it is an ideal target for MM immunotherapy. Several clinical trials evaluating different BCMA-targeting CAR constructs have been initiated and early results are very promising. However, in this rapidly developing clinical landscape, the ultimate role of BCMA-specific CAR-T cell therapy remains unclear. In this review, we will summarize currently available clinical data on BCMA-directed CAR-T cells and discuss potential future perspective for this promising treatment approach in MM.     

DC-CIK细胞过继免疫治疗联合XELOX方案化疗对进展期结直肠癌患者的疗效分析

目的探讨树突状细胞-细胞因子诱导杀伤细胞(DC-CIK)细胞过继免疫治疗联合奥沙利铂+卡培他滨(XELOX)方案化疗对进展期结直肠癌(CRC)患者的疗效。方法将104例进展期CRC患者按随机数字表法分为观察组和对照组各52例,观察组行DC-CIK联合化疗,对照组行化疗。检测患者治疗前后血清糖类抗原242(CA242)、糖类抗原19-9(CA19-9)、癌胚抗原(CEA)、T淋巴细胞亚群[自然杀伤性T(NKT)细胞、自然杀伤(NK)细胞、CD4^+、CD3^+、CD8^+]及白细胞介素-6(IL-6)、白细胞介素-2(IL-2)、干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)水平,评价患者近期疗效。结果观察组患者的缓解率(RR)为61.54%(32/52),疾病控制率(DCR)为75.00%(39/52),分别高于对照组的40.38%(21/52)和55.77%(29/52),差异均有统计学意义(P<0.05)。治疗后,两组患者血清CEA水平均低于治疗前,差异均有统计学意义(P<0.05);治疗后,观察组患者血清IL-2、IFN-γ、TNF-α、CD4^+、CD3^+、CD8^+及NKT细胞水平均高于对照组,差异均有统计学意义(P<0.05)。结论 CRC患者行DC-CIK细胞过继免疫治疗联合XELOX方案化疗可改善机体免疫功能,弥补单纯化疗所造成的免疫抑制,为安全有效的疗法。     

肿瘤生物治疗开启黑素瘤临床治疗新时代

恶性黑素瘤是一类进展快预后差的恶性肿瘤,对传统放化疗均不敏感,晚期患者5年生存率不足5%。随着单克隆抗体、小分子化合物、过继性免疫细胞和溶瘤病毒等生物治疗技术的研发,肿瘤生物治疗为恶性黑素瘤的临床治疗开启了一个新的时代。2011年到2014年,CTLA-4单抗Ipilimumab,PD-1单抗Pembrolizumab、Nivolumab,BRAF抑制剂Vemurafinib、Dabrafinib和MEK抑制剂Trametinib等相继获得FDA批准用于治疗晚期黑素瘤患者,同时多种自体免疫细胞疗法如TIL、CAR-T,以及溶瘤病毒T-VEC等也都在其各自的临床试验中获得了可靠的疗效证据。肿瘤生物治疗以其独特的治疗优势,打破了恶性黑素瘤临床研究近50年的沉寂。然而,我国恶性黑素瘤的生物治疗临床研究尚处于起步阶段,多种生物治疗技术在中国的推广仍需进一步的临床佐证。但随着研究的不断深入,尤其是细胞免疫学、分子生物学和肿瘤遗传学等学科的发展和融合,越来越多的生物治疗方法将逐渐应用于临床,造福于更多的恶性黑素瘤患者。     

非小细胞肺癌过继细胞免疫治疗的研究进展

目前非小细胞肺癌发病率高,预后差,需要研究新的治疗方法。肿瘤的发生、发展和转移与人体免疫系统密切相关,过继输注免疫细胞是免疫治疗的一种方法,能够显著提高机体免疫系统的抗肿瘤效应。过继输注的免疫细胞多样,如 LAK、TIL、NK、γδ T、CIK、CTL 等,通过输注不同的免疫细胞或联合其他治疗介导靶细胞凋亡,可增强抗肿瘤效应。本文介绍各种免疫细胞的抗肿瘤的机制并回顾近几年治疗非小细胞肺癌的相关试验研究。     

DC-CIK细胞联合化疗治疗广泛期小细胞肺癌的临床疗效

目的:评价DC-CIK细胞联合化疗对广泛期小细胞肺癌(small cell lung cancer,SCLC)的有效性及安全性.方法:收集2012年2月至2015年1月就诊于上海东方肝胆外科医院的15例广泛期SCLC患者,采用EP方案(依托泊甙+顺铂)化疗结束后3～5d进行DC-CIK细胞治疗,每21 d为一周期,观察联合治疗前后患者外周血中T细胞亚群的变化、临床疗效、生活质量和不良反应等.结果:15例患者均可评价疗效,获CR 3例、PR 8例、SD 3例、PD 1例、RR为73.3％,DCR为93.3％,PFS为6.8个月,OS为14.9个月,其中2例获CR的患者OS分别为29.1个月、37.2个月,2年生存率达13.3％;联合治疗后较治疗前CD4+ CD25+ CD127-调节性T细胞、CD4+T细胞减少[(5.42±0.70)％ vs (6.16±0.77)％,P＜0.05;(28.74±1.92)％ vs (33.06±2.69)％,P＜0.05],CD3+ CD56+细胞因子诱导的杀伤细胞、CD8 +T细胞增加[(5.58±0.94)％ vs (4.94±0.77)％,P ＜0.05;(43.26±3.87)％ vs(39.92±2.78)％,P＜0.01],CD4 +/CD8+T细胞比值明显下降[(0.67±0.09)％vs (0.83±0.10)％,P＜0.01];治疗后患者KPS评分提高20分2例、10分9例,有效率为73.3％,不良反应主要表现为轻中度骨髓抑制、胃肠道反应,给予对症处理后可恢复正常.结论:DC-CIK细胞联合化疗可提高广泛期SCLC患者的临床缓解率,延长生存期,明显改善生活质量,增强免疫功能,安全有效.     

Soluble γc receptor attenuates anti‐tumor responses of CD8+ T cells in T cell immunotherapy

Previous studies have shown that soluble common γ‐chain (sγc) modulates CD4⁺ T cell immunity with antagonistic functions in γc cytokine signaling. However, the role of sγc in functional properties of effector CD8⁺ T cells has not been fully defined. In this study, we report a new mechanism by which the anti‐tumor activity of mouse CD8⁺ T cells is suppressed in sγc of their own producing. While sγc significantly inhibits cytotoxicity of CD8⁺ T cells, blocking sγc production by genetic modification leads to potentiated effector function of CD8⁺ T cells, establishing persistent CD8⁺ T cells. This is due to the modulation of IL‐2 and IL‐15 signaling, which is required for expansion and survival of CD8⁺ T cells as well as for optimal cytotoxic activity. More efficient management of tumor growth was achieved by an adoptive transfer of sγc‐deficient CD8⁺ T cells than that of wild‐type or sγc‐overexpressing CD8⁺ T cells. Blocking of IL‐2 and IL‐15 signaling by sγc attenuates the capacity of CD8⁺ T cells to mount an optimal response to the tumor, with both quantitative and qualitative effects on antigen‐specific CD8⁺ T cells. These results could have a critical implication for the generation and survival of optimal effector T cells for adoptive immunotherapy of cancer.     

肿瘤抗原多肽诱导的CD8~+CTL的体内外抗瘤效应

利用海绵移植模型,将Friend小鼠白血病病毒gag编码的二个多肽CCLCLTVFL(gPr80 gag 85-93)和RSPTNLAKV(Pr65 gag P30 131-139)分别注入正常或经FBL-3肿瘤细胞免疫过的C57BL/6小鼠皮下的植入海绵中,10天后,收集海绵中浸润淋巴细胞,在体外用免疫肽周期性刺激培养扩增。实验结果表明:用多肽体内免疫正常或经FBL-3免疫的B6小鼠均能诱导出对免疫多肽特异的CD8~ CTL。其中,P85-93多肽诱导的CD8~ CTL除能杀伤该多肽致敏的同系靶细胞外,还能特异性杀伤FBL-3瘤细胞.抗原多肽诱导的CD8~ CTL在体外经用免疫多肽致敏的同系小鼠脾细胞作周期性刺激并在低浓度IL-2的条件下培养,可长期生长并大量扩增。用体外培养扩增的P85-93多肽特异性CD8~ CTL过继转输治疗FBL-3荷瘤小鼠,能有效治愈FBL-3白血病荷瘤小鼠。上述结果表明,用肿瘤抗原多肽替代肿瘤细胞体内免疫动物可诱导特异性抗瘤效应的CD8~ CTL,并可在体外扩增用于肿瘤过继性免疫治疗。     

Peptide-specific CD8+ T-cell evolution in vivo: response to peptide vaccination with Melan-A/MART-1

Monitoring of CD8+ T-cell responses in cancer patients during peptide vaccination is essential to provide useful surrogate markers and to demonstrate vaccine efficacy. We have longitudinally followed CD8+ T-cell responses in 3 melanoma patients who were immunized with peptides derived from Melan-A/MART-1. Recombinant HLA-A2 tetramers loaded with the naturally presented Melan-A/MART-1 nonamer peptide (AAGIGILTV) and the Melan-A/MART-1 analog (ELAGIGILTV) were used in combination with phenotypical analysis for different T-cell subsets including naive T cells, effector T cells, "true memory" T cells and "memory effector" T cells, based on CD45RA/RO and CCR7-expression. At least in a single patient, T cells binding to the AAGIGILTV epitope were detected in naive, precursor (CD45RA+/CCR7+) CD8+ T cells, and CD8+ T cells binding to the analog ELAGIGILTV peptide were identified in the terminally differentiated (CD45RA+/CCR7-) T-cell subset. Molecular and functional analysis of tetramer-binding T cells revealed that the T-cell receptor (TCR) repertoire was oligo/polyclonal in AAGIGILTV-reactive T cells, but different and restricted to a few TCR clonotypes in ELAGIGILTV-reactive T cells prior to vaccination. The TCR repertoire reactive with Melan-A/MART-1 peptide epitopes was broadened during vaccination and exhibited a different profile of cytokine release after specific stimulation: tetramer-binding T cells from 2/3 patients secreted granulocyte/macrophage colony-stimulating factor (GM-CSF) and interferon-gamma but not interleukin-2 (IL-2) in response to Melan-A/MART-1 peptides. In the third patient, tetramer-binding T cells secreted IL-2 exclusively. Our results show that T-cell responses to peptide vaccination consist of different T-cell subsets associated with different effector functions. Complementary analysis for TCR CDR3 and cytokine profiles may be useful to define the most effective CD8+ T-cell population induced by peptide vaccination.     

Cancer treatment by photodynamic therapy combined with adoptive immunotherapy using genetically altered natural killer cell line.

Adoptive immunotherapy mediated by human natural killer (NK) cell line genetically altered to produce interleukin-2 (NK92MI) was evaluated as adjuvant to photodynamic therapy (PDT) of subcutaneous tumors. The combined effect of these two modalities was first examined with SiHa tumors (human cervical squamous cell carcinoma) growing in NOD-scid mice. The most effective protocol for NK92MI cell transfer in conjunction with PDT mediated by photosensitizer mTHPC was the injection of 5 x 10(7) cells (peritumoral or intravenous) given immediately after PDT, which produced a marked improvement in the therapeutic outcome compared with the effect of PDT alone. The same protocol was tested with HT-29 tumor model (human colorectal adenocarcinoma) xenografted in NOD-scid mice. The results demonstrate that the adoptive immunotherapy with NK92MI cells (which when used alone were not effective in controlling tumor growth) significantly improved the cures of PDT-treated HT-29 tumors, whereas such benefit was not observed with the parental cell line NK92 (not producing interleukin-2). Flow cytometry-based analysis revealed a higher percentage of p.t. injected NK92MI cells in PDT-treated than in non-treated HT-29 tumors. Further investigation showed that the NK92MI cell-based adoptive immunotherapy is also a highly effective adjuvant for PDT treatment of murine EMT6 tumors growing in immunocompetent syngeneic BALB/c mice. This result diminishes the concern that adoptively transferred NK92MI cells may be rendered ineffective by an allogenic reaction of the host. The findings of this study suggest that advanced protocols of NK cell-based adoptive immunotherapy can be developed as efficient adjuvants to PDT used for the treatment of solid malignant tumors.     

Potential use of T cell receptor genes to modify hematopoietic stem cells for the gene therapy of cancer

The purpose of this review is to illustrate some of the technical and biological hurdles that need to be addressed when developing new gene therapy based clinical trials. Gene transfer approaches can be used to “mark” cells to monitor their persistence in vivo in patients, to protect cells from toxic chemotherapeutic agents, correct a genetic defect within the target cell, or to confer a novel function on the target cell. Selection of the most suitable vector for gene transfer depends upon a number of factors such as the target cell itself and whether gene expression needs to be sustained or transient. The TCR gene transfer approach described here represents one innovative strategy being pursued as a potential therapy for metastatic melanoma. Tumor reactive T cells can be isolated from the tumor infiltrating lymphocytes (TIL) of melanoma patients. A retroviral vector has been constructed containing the T cell receptor (TCR) α and β chain genes from a MART-1_(27-35)-specific T cell clone (TIL 5). Jurkat cells transduced with this virus specifically release cytokine in response to MART-1_(27-35) peptide pulsed T2 cells, showing that the virus can mediate expression of a functional TCR. HLA-A2 transgenic mice are being used to examine whether transduced bone marrow progenitor cells will differentiatein vivo into mature CD8⁺ T cells expressing the MART-1_(27-35)-specific TCR. Expression of the human TCR α and β chain genes has been detected by RT-PCR in the peripheral blood of HLA-A2 transgenic mice reconstituted with transduced mouse bone marrow. Expression of the TIL 5 TCR genes in the peripheral blood of these mice was maintained for greater than 40 weeks after bone marrow reconstitution. TIL 5 TCR gene expression was also maintained following transfer of bone marrow from mice previously reconstituted with transduced bone marrow to secondary mouse recipients, suggesting that a pluripotent progenitor or lymphocyte progenitor cell has been transduced.     

Immunotherapies targeting neoantigens are effective in PD-1 blockade-resistant tumors

Only a small fraction of tumor-infiltrating lymphocytes can specifically recognize and attack cancer cells in PD-1/PD-L1 blockade therapy. Here, we investigate approaches to expand the neoantigen-specific CD8⁺ T cells to overcome the difficulties in treating PD-1/PD-L1 blockade-resistant tumors. Mutation-associated neoepitopes of murine nonsmall cell lung cancer ASB-XIV were estimated by whole-exome and RNA sequencing and predicted by MHC-I binding affinity (FPKM >1) in silico. Using ASB-XIV-specific CD8⁺ T cells, we screened a panel of 257 neoepitope peptides derived from ASB-XIV missense and indel mutations. Mutated Phf3 peptide (mPhf3) was successfully identified as an immunogenic neoepitope. Prophylactic mPhf3-DC vaccination inhibited ASB-XIV tumor growth through CD8⁺ T cell-mediated antitumor immunity. Combining the mPhf3-DC vaccine and anti-PD-1 treatment elicited robust antitumor activity through the induction of mPhf3-specific CD8⁺ T cells in the tumor microenvironment. Furthermore, the adoptive transfer of mPhf3-specific CD8⁺ T cells eradicated ASB-XIV tumors. Likewise, the combination of mutated Cdt1 peptide (mCdt1)-DC vaccine and anti-PD-1 treatment or adoptive transfer of mCdt1-specific CD8⁺ T cells also led to significant regression of PD-1 blockade-resistant murine gastric YTN16 tumors. In conclusion, a novel immunogenic neoepitope of ASB-XIV was identified for immunotherapy targeting neoantigens. Identification of immunogenic neoantigens can extend the therapeutic strategies by increasing the frequency of neoantigen-specific T cells, even for PD-1/PD-L1 blockade-resistant tumors.     

Peripheral γδ T-lymphocytes as an innovative tool in immunotherapy for metastatic renal cell carcinoma

Renal cell carcinoma represents 3% of solid malignancies in adults and nephrectomy remains the main treatment. Failure of conventional approaches for patients presenting with advanced disease has prompted the exploration of new strategies. This review describes the potentialuse of peripheral γδ (Vγ9Vδ2) T-cells in metastatic renal cell carcinoma. This peripheral lymphocyte population from the innate immune system has demonstrated an in vitro antitumor cytotoxicity against primary or established renal cell lines. Moreover, these Vγ9Vδ2 lymphocytes undergo a rapid and extensive expansion in vitro as well as in vivo upon stimulation with a synthetic potent agonist, the bromohydrin pyrophosphate molecule. Preclinical results obtained on specific in vitro amplification of Vγ9Vδ2 T-cells by bromohydrin pyrophosphate in renal cell carcinoma patients are presented in this review, while Phase I clinical trials are currently running. As there is growing evidence for the low efficiency of monotherapy in cancer patients, innovative approaches combining immunomodulatory γδ agonists with classic chemotherapies or administration of antiangiogenic agents are discussed.