ADMINISTRATION OF EBV-SPECIFIC CYTOTOXIC T-LYMPHOCYTES FOLLOWING CD45 ANTIBOD

CD45 抗体后给予 EBV 特异性细胞毒性 T 淋巴细胞

• 批准号: 7605935
• 负责人: HELEN E HESLOP
• 金额: $ 1.05万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-15 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7605935
• 关键词: Adoptive Immunotherapy; Adoptive Transfer; Adult; Adverse effects; Allogenic; Animal Experiments; Animals; Antibodies; Antigen Targeting; Antigens; Apoptosis; Apoptotic; Area; Autologous; B-Lymphocytes; Biological Assay; Blood; Bulky Disease; Bypass; CD4 Positive T Lymphocytes; CD45 Antigens; CD8B1 gene; Cell Count; Cells; Characteristics; China; Clinical; Colon Carcinoma; Computer Retrieval of Information on Scientific Projects Database; Count; Cyclophosphamide/Fludarabine; Cytomegalovirus; Cytotoxic agent; Data; Disease; Disease regression; Disease remission; Disease-Free Survival; Disruption; Distant; Donor Lymphocyte Infusion; Dose; Drug usage; EBV-Specific Cytotoxic T-Lymphocyte; EBV-associated malignancy; End Point; Engraftment; Environmental Exposure; Epitopes; Epstein Barr Nuclear Antigen 3; Failure; Funding; Genetic Predisposition to Disease; Geographic Locations; Grant; Half-Life; Helper-Inducer T-Lymphocyte; Hematopoietic; Hematopoietic stem cells; Histocompatibility Antigens Class I; Homeostasis; Hour; Human; Human Herpesvirus 4; Human Papillomavirus; Image; Immune; Immune response; Immune system; Immunocompromised Host; Immunotherapeutic agent; Immunotherapy; In complete remission; Incidence; Infection; Infusion procedures; Institution; LMP1; Life; Ligands; Living Wills; Lymphocyte; Lymphocyte Count; Lymphocyte Depletion; Lymphoid; Lymphoma; Lymphopenia; Lymphoproliferative Disorders; Lytic; Malignant Neoplasms; Malignant neoplasm of nasopharynx; Marrow; Mature T-Lymphocyte; Measurable; Measures; Mediating; Membrane Proteins; Memory; Messenger RNA; Methods; Monitor; Monoclonal Antibodies; Morbidity - disease rate; Mus; Myelogenous; Nasopharynx Carcinoma; Natural regeneration; Neutropenia; Nuclear Pore Complex; Numbers; Organ; Outcome; PTPRC gene; Pathogenesis; Patients; Peptides; Peripheral; Phase I Clinical Trials; Phenotype; Plasma; Play; Prevention; Process; Proliferating; Proteins; Publishing; Radiation; Radiation therapy; Rate; Recovery; Recurrent disease; Refractory; Regulation; Relapse; Research; Research Personnel; Residual Tumors; Residual state; Resources; Risk; Role; Safety; Site; Smoke; Source; Spleen; Squamous Cell; Staging; Staining method; Stains; Stem cell transplant; Stem cells; Stimulus; Symptoms; System; T cell regulation; T memory cell; T-Cell Depletion; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Subsets; TAP1 gene; TAP2 gene; Techniques; Tetanus Toxoid; Thinking; Toxic effect; Transplant Recipients; Treatment Protocols; Treatment outcome; Undifferentiated; United States National Institutes of Health; Viral load measurement; Virus; Virus Latency; Work; abstracting; chemotherapy; cytokine; cytotoxic; day; dosage; enzyme linked immunospot assay; experience; human study; improved; in vivo; leukemia; lymph nodes; man; melanoma; men; mortality; neoplastic cell; novel; partial response; peripheral blood; prevent; progenitor; prophylactic; reconstitution; response; restoration; size; success; tumor; tumor growth

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT I. HYPOTHESIS We have previously used adoptively transferred EBV-specific CTL to prevent and treat EBV-lymphoproliferative disease after stem cell transplant1,2. Recently we have investigated whether the same approach could be of value to treatment for EBV+ Nasopharyngeal cancer(NPC)3. We have been successful in generating autologous EBV-specific CTL lines for NPC patients using a standardized method. Adoptive transfer of EBV-specific CTLs has proven safe in all 10 NPC patients treated. In six patients with refractory or relapsed disease, CTL infusion resulted in 1 complete response, 2 partial responses and stabilization of disease for >6 months in 1 case with 2 patients too early to evaluate. Four patients who were treated in remission have remained disease free for >5->13 months. The above results are promising and indicate the potential anti-tumor activity of EBV-specific CTLs in this patient group. However, not all patients with bulky disease responded completely. We believe this failure was due in part to the failure of infused T lymphocytes to undergo adequate expansion in vivo. The size of the T cell compartment is maintained at a steady state by a number of potent homeostatic mechanisms4. If a host is made lymphopenic, mature T cells will proliferate to restore the steady state5. Hence, in the recipients of T cell depleted stem cell transplants we treated previously, we have found that infused EBV specific T cells expand by 4 logs or more1,6. In contrast, when EBV specific T cells are given to NPC patients, in whom the T cell compartment is already replete, expansion of adoptively transferred T lymphocytes is lower, by 2 orders of magnitude or more. Published data suggest that in vivo depletion of T cells with cytotoxic drugs, as in stem cell transplantation, can permit massive expansion of subsequently infused T cells with anti-cancer activity, since under those circumstances homeostatic mechanisms work to restore lymphoid compartments7. We now propose to take advantage of these homeostatic mechanisms in patients with advanced or refractory NPC. We will deplete their lymphoid compartment using short-lived CD45 monoclonal antibodies (MAbs), which we have shown (in mouse and in man) can profoundly deplete lymphocytes in peripheral blood and lymphoid organs, whilst sparing hematopoietic progenitor cells8,9. Subsequent adoptive transfer of EBV-specific CTL post MAb induced T cell depletion should result in expansion of the infused cells to restore the T cell compartment. Moreover, the expanded cells should enter and function unimpeded in tumor sites depleted of regulatory T cells. II. SPECIFIC AIMS Aim 1) To perform a phase I study of EBV-specific CTL infusion before and after administration of CD45 monoclonal antibody in patients with NPC. Primary clinical endpoints of this study will be safety and hematopoietic and immune reconstitution. In our previous studies in animal and humans, lytic CD45 MAbs safely depleted lymphocytes of all lineages, but spared hematopoietic progenitor cells, which have a low level of target antigen expression. The antibodies have a half-life of circa 8 hours and therefore cause only transient neutropenia with no measurable stem cell toxicity. The degree of T cell depletion will be determined by total and differential T cell counts and phenotyping. We plan to monitor hematopoietic and immune reconstitution by measuring responses to recall antigens such as cytomegalovirus (CMV) and tetanus toxoid (TT) using tetramers and ELISPOT. The clinical implications of transient lymphopenia in these patients will be monitored with emphasis on the occurrence of potential side effects such as infection. Aim 2) To analyze the effect of CD45-mediated host T cell depletion on the expansion, persistence and anti-tumor effector function of subsequently infused EBV-specific CTL. This aim will be achieved by comparing expansion, persistence and function of infused T cells given to the same patient before and after CD45 MAb treatment. We will use tetramer staining and functional ELISPOT assays to monitor the expansion and persistence of adoptive transferred EBV-specific CTL. Anti-tumor effects will be evaluated by measuring cell-free EBV-DNA in plasma, tumor size on imaging and clinical outcome III. BACKGROUND AND SIGNIFICANCE 1. Nasopharyngeal carcinoma 1.1 NPC treatment and outcome. The incidence of nasopharyngeal carcinoma varies widely with geographical location, with an incidence as high as 50 per 100,000 men in Southern China and less than 1 per 100,000 adults in low incidence areas including the USA10. NPC is a radiosensitive tumor, and by modern imaging and radiation techniques, local control rates of greater than 80% are obtained11. Despite this, distant failures remain the major problem in patients with loco-regional bulky disease, which is the most common form at presentation due to the lack of early symptoms. At present, radiotherapy combined with induction or concurrent chemotherapy results in a 5-year survival of 55-70% in patients with advanced stage disease. However, 40-50% of patients relapse12 and the treatment-related morbidity and mortality of the current regimens are of major concern2. It is therefore desirable to develop novel therapies that could improve disease-free survival in relapsed/refractory patients and which might ultimately reduce the incidence of long-term treatment related complications in all patients. 1.2 Nasopharyngeal carcinoma and Epstein-Barr virus. Multiple factors including EBV exposure, environmental triggers and genetic susceptibility are thought to play a role in the pathogenesis of NPC13. EBV has been detected in virtually all cases of undifferentiated non-keratinizing NPC14 and in a proportion of squamous cell NPC17. The latter represents a more heterogeneous group of tumors, in which other co-factors such as smoking and human papilloma virus (HPV) contribute to the pathogenic process15. The strong association with EBV makes NPC an attractive candidate for immunotherapeutic strategies. Although EBV positive NPC cells lack the expression of the immunodominant EBNA3 antigens, LMP 1 is expressed in 65% of NPC tumors and transcriptional analysis has shown expression of LMP2 mRNA in the majority of tumors16. Therefore, these membrane proteins provide target antigens for immunotherapeutic strategies. Importantly, T lymphocytes specific for LMP2 and to a lesser extent for LMP1 are present in the peripheral blood of NPC patients17;18 and could therefore potentially be activated and expanded for immunotherapeutic approaches. 2. Immunotherapy with antigen-specific T cells. 2.1 Successes. Immunotherapy with T cells has been most successful in stem cell transplant recipients, for whom the normal marrow donors have been used as the source of T cells. Adoptive immunotherapy with donor lymphocyte infusions (DLI) after allogeneic hematopoietic stem cell transplant (HSCT) has provided an effective means of augmenting the graft versus leukemia response to eliminate residual disease and for the treatment of EBV-associated lymphoproliferative diseases occurring after HSCT. However, DLI are associated with a high risk of GvHD19,20,20. Selectively expanded CMV and EBV-specific CTL have successfully restored immune responses and prevented diseases associated with these viruses without causing GvHD2,21. Infusion of CMV-specific CD4+ and CD8+ clones demonstrated that the survival of the CD8+ CTL was dependent on the presence of CD4+ CMV-specific "helper" T cells. Similarly, polyclonal EBV-specific CTL, containing both CD4+ and CD8+ T cells survived for up to eighty-six months after infusion and were able to reduce the high virus load observed in about 20% of patients1,2. EBV-CTL also appeared to prevent progression to EBV-lymphoma, since no patient who received prophylactic CTL developed this malignancy, compared to 11.5% of controls2. Further, five of six patients who received CTL as treatment for overt lymphoma achieved complete remissions. In the patient who failed to respond, the tumor was transformed with a virus that had deleted the two CTL epitopes for which the donor CTL line was specific22. 2.2 Rationale for immunotherapy of Nasopharyngeal carcinoma with EBV-specific CTLs. A major advantage offered by CTL therapy is its lack of toxicity. If CTL are effective in the treatment of relapsed disease, they could subsequently be used as part of front line therapy for nasopharyngeal carcinoma and allow a reduction in radiotherapy and chemotherapy dosage and hence in long-term sequelae. Since adoptively transferred CTL have proved long-lived and effective in the prevention and treatment of EBV-associated PTLD in HSCT recipients, they have the potential to be beneficial in patients with other EBV-associated malignancies that express viral latency proteins that are potential CTL targets. Importantly, NPC tumor cells express MHC class I molecules as well as the peptide transporters TAP1 and TAP2, and are therefore capable of processing and presenting endogenously synthesized protein in the context of HLA class I MHC molecules, for immune recognition by CTL23. 3. Disruption of lymphoid homeostasis to facilitate expansion and engraftment of infused CTLs. 3.1 Rationale for T cell depletion prior to CTL infusion. Lymphocyte numbers and subset composition are maintained at stable levels in healthy adults. Perturbations of cell numbers due to infection, loss of cells, or other influences are restored to steady-state levels by poorly understood homeostatic mechanisms. Each lymphoid compartment maintains homeostasis more or less independently, so that in T cell deficient mice, B cell numbers remain the same as in normal mice. Similarly, regulation of the nanve T cell subset, which derives from thymic proliferation and maintains T cell receptor diversity, is independent of memory T cell regulation, which is maintained by antigen-independent as well as antigen-dependent mechanisms24. After T cells are depleted, lymphoid reconstitution with a fully diversified T cell receptor repertoire is dependent on thymopoiesis, but this continues at a steady rate, regardless of the size of the lymphoid compartment. Conversely, expansion of memory T cells is the major source of peripheral repopulation25,26. Regulation of lymphocyte numbers may be controlled by cytokines or ligands, by counting mechanisms, or by the availability of "space". While these mechanisms are bypassed during immune responses, with increases in the size of spleen, lymph nodes and blood, there is a rapid return to a steady state when antigen stimulation subsides. Expansion of infused T cells is likely to be limited in a steady state situation. In the context of a T cell deficit however, infused cells have been shown to proliferate rapidly to restore steady situation5. Animal experiments provide us with important rules regulating the restoration of homeostasis when confronted with a lymphopenic situation. First, T cell proliferation to restore a deficit is not dependent on an antigen stimulus, although activated T cells have a proliferative advantage which is increased in the presence of antigen24. Second, once the peripheral pool is replenished, further expansion is curtailed, which prevents unlimited expansion of infused lymphocytes. Third, rapid repopulation of the peripheral pool can be achieved with a single infusion of mature T lymphocytes, even using a small number of cells. Conversely, in case of high dose lymphocyte infusion, steady state is achieved by apoptotic rather than proliferative mechanisms. Since entry into the memory pool can only be established by proliferation27, administration of an appropriate cell number may be crucial to allow for in vivo expansion and thus long-term anti-tumor protection. Finally, additional poorly understood mechanisms maintain peripheral diversity and prevent an antigen-driven progression to oligoclonality. In sublethally irradiated lymphopenic mice, polyclonal but not clonal autologous T cells prevented tumor growth when the animals were challenged with melanoma or colon carcinoma cells28. Thus homeostasis by proliferation may explain why infusions of small numbers of polyclonal EBV-specific CTLs expanded by 4 logs after T cell-depleted BMT, and persisted for up to eighty-six months post infusion: the T cell compartment was depleted; the infused T cells were activated; and EBV antigens were present. By contrast, homeostasis by apoptosis may explain why large numbers of clonal T cells infused into T cell depleted patients rapidly disappear after infusion and never enter the memory pool29. The above described mechanisms of lymphocyte homeostasis imply that the administration of a small number of EBV-specific T cells post lymphocyte depletion may allow for significant in vivo expansion and thereby improve their anti-tumor function. Furthermore, depletion of tumor-infiltrating T cells with an inhibitory effect on the function of adoptively transferred CTL may be an additional advantageous effect of lymphocyte depletion prior to CTL infusion. This secondary gain is further discussed in the rationale of Aim 2. Lymphoid depletion as a strategy to create space for the expansion of adoptively transferred cells and to facilitate their entry into the tumor site has already shown evidence of success. When melanoma patients received cyclophosphamide and fludarabine prior to the adoptive transfer of large numbers of highly-activated melanoma-specific tumor infiltrating T cells (TIL), repopulation and proliferation of the transferred cells was observed, as well as regression of metastatic melanoma7. However, some patients remained profoundly immunocompromised and failed to regenerate an effective immune system from these infused TILs, their expansion notwithstanding. This poor immune reconstitution resulted in part from the extensive and non-specific destruction of the resident immune system by the cytotoxic drugs used. A method of T cell depletion with less systemic toxicity, particularly to the myeloid system, may allow in vivo expansion of infused CTL and recovery of the residual lymphoid compartment. CD45 MAb, which targets a common leucocyte antigen, profoundly depletes the lymphocyte compartment for approximately 30 days while the myeloid system regenerates within 72 hours. In combination with its short half-life (about 8 hours), which allows for CTL infusion shortly after MAb administration, these characteristics make CD45 Mab a potentially safe and effective method of lymphodepletion. Our experience with CD45 MAbs in murine and human studies is discussed in section C(7). 4. Summary and implications We hypothesize that the anti-tumor activity of EBV-specific CTL infused into patients with EBV+ NPC can be increased dramatically by prior depletion of the T cell compartment to allow for expansion of infused tumor-specific T cells. We believe that CD45 MAbs will safely and substantially deplete normal T cells, and their short half-life will allow infusion of EBV-specific CTL shortly after CD45 MAb administration, enabling their expansion prior to the recovery of endogenous T cells. This study will provide important information on the ability of MAb mediated T cell depletion to enhance the proliferation of adoptively transferred, tumor specific T cells.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，可以在其他清晰的条目中表示。列出的机构是 对于中心，这不一定是调查员的机构。 抽象的 I.假设 我们以前已经使用了过继转移的EBV特异性CTL来预防和治疗干细胞移植后1,2后EBV淋巴增生性疾病。最近，我们研究了EBV+鼻咽癌（NPC）3的治疗方法是否具有价值。我们已经成功地使用标准化方法为NPC患者生成了自体EBV特异性CTL系。在所有10名NPC患者中，EBV特异性CTL的收养转移均被证明是安全的。在六名难治性或复发性疾病的患者中，CTL输注导致1个完全反应，2个部分反应和疾病稳定在1例中> 6个月，有2例患者过早无法评估。在缓解中接受治疗的四名患者持续> 5-> 13个月。 上述结果是有希望的，表明该患者组EBV特异性CTL的潜在抗肿瘤活性。但是，并非所有笨重的患者都做出了完全反应。我们认为，这种失败的部分原因是注入的T淋巴细胞在体内进行足够的扩张。 T细胞室的大小通过多种有效的稳态机制维持在稳定状态。如果使宿主成为淋巴细胞减少症，则成熟的T细胞将增殖以恢复稳态5。因此，在我们先前治疗的T细胞耗尽的干细胞移植物的受体中，我们发现注入的EBV特异性T细胞膨胀了4个或1,6。相反，当将EBV特异性T细胞提供给NPC患者（T细胞室已经充满的NPC患者）时，采用转移的T淋巴细胞的扩展较低，降低了2个数量级。公开的数据表明，与干细胞移植一样，用细胞毒性药物在体内耗竭可以允许大量扩大随后注入的T细胞具有抗癌活性，因为在这种情况下，稳态机制可恢复淋巴结室7。现在，我们建议在晚期或难治性NPC患者中利用这些稳态机制。我们将使用短寿命的CD45单克隆抗体（MAB）耗尽其淋巴区室，我们（在小鼠和人中）表明，这些抗体可以在外周血和淋巴样器官中深刻耗尽淋巴细胞，并保留造血性象征性递质细胞8.9。随后的EBV特异性CTL后MAB诱导的T细胞耗竭的继发性转移应导致注入的细胞扩大以恢复T细胞室。此外，扩展的细胞应在调节性T细胞中耗尽的肿瘤部位中不受阻碍的输入和功能。 ii。 具体目标 目标1）在施用NPC患者的CD45单克隆抗体之前和之后，对EBV特异性CTL输注进行I期研究。这项研究的主要临床终点将是安全，造血和免疫重建。在我们先前对动物和人类的研究中，裂解的CD45 mAB安全地耗尽了所有谱系的淋巴细胞，但含有较低靶向抗原表达水平的造血祖细胞。抗体的半衰期约为8小时，因此仅引起瞬时中性粒细胞减少症，而没有可测量的干细胞毒性。 T细胞耗竭的程度将由总T细胞计数和表型确定。我们计划通过测量对抗原抗原（例如巨细胞病毒（CMV）和破四型毒素（TT）的回忆反应来监测造血和免疫重建，使用四聚体和ELISPOT。这些患者中短暂性淋巴细胞减少症的临床意义将受到监测，重点是引起潜在副作用（例如感染）。 目标2）分析CD45介导的宿主T细胞耗竭对随后注入EBV特异性CTL的膨胀，持久性和抗肿瘤效应功能的影响。通过比较CD45 mAb治疗前后给予同一患者的膨胀，持久性和功能来实现此目标。我们将使用四聚体染色和功能性ELISPOT分析来监视继发性转移的EBV特异性CTL的膨胀和持久性。将通过测量血浆中无细胞的EBV-DNA，肿瘤大小和临床结局来评估抗肿瘤效应 iii。 背景和意义 1。鼻咽癌 1.1 NPC治疗和结果。鼻咽癌的发病率在地理位置差异很大，在中国南部，发病率高达每10万人50人，在包括USA10在内的低发地区地区，每10万名成年人不到每10万名成年人。 NPC是一种放射敏感性肿瘤，通过现代成像和辐射技术，获得了大于80％的局部控制速率11。尽管如此，遥远的失败仍然是现场区域笨重疾病的患者的主要问题，由于缺乏早期症状，这是表现最常见的形式。目前，放射疗法与诱导或同时化疗相结合导致晚期疾病患者的5年生存率为55-70％。然而，40％至50％的患者复发12和与治疗相关的发病率和当前方案的死亡率是主要关注的2。因此，需要开发新的疗法，可以改善复发/难治性患者中无疾病的生存率，并且最终可能会减少所有患者长期治疗相关并发症的发生率。 1.2鼻咽癌和爱泼斯坦 - 巴尔病毒。 EBV暴露，环境触发因素和遗传敏感性在内的多种因素被认为在NPC13的发病机理中起作用。在几乎所有未分化的非能型NPC14和鳞状细胞NPC17的比例的情况下，已经检测到EBV。后者代表了一种更异质的肿瘤，其中其他共同因素（如吸烟和人乳头瘤病毒（HPV））有助于致病过程15。与EBV的牢固联系使NPC成为免疫治疗策略的有吸引力的候选人。尽管EBV阳性NPC细胞缺乏免疫主导EBNA3抗原的表达，但在65％的NPC肿瘤中表达LMP 1，转录分析显示了大多数肿瘤中LMP2 mRNA的表达16。因此，这些膜蛋白为免疫治疗策略提供了靶抗原。重要的是，NPC患者的外周血中存在于LMP2和LMP1较小程度的T淋巴细胞17; 18; 18，因此有可能被激活并扩展以进行免疫治疗方法。 2。与抗原特异性T细胞的免疫疗法。 2.1成功。用T细胞的免疫疗法在干细胞移植受者中最成功，为此，正常的骨髓供体被用作T细胞的来源。同种异性造血干细胞移植（HSCT）后，用供体淋巴细胞输注（DLI）采用免疫疗法（DLI）提供了一种有效的方法来增加移植物与白血病反应，以消除残留疾病，以消除EBV相关淋巴细胞增多症的治疗。但是，DLI与GVHD19,20,20的高风险有关。选择性扩展的CMV和EBV特异性CTL已成功恢复免疫反应，并预防与这些病毒相关的疾病，而不会引起GVHD2,21。输注CMV特异性CD4+和CD8+克隆表明，CD8+ CTL的存活取决于CD4+ CMV特异性“助手” T细胞的存在。同样，输注后含有CD4+和CD8+ T细胞的多克隆EBV特异性CTL存活长达86个月，并且能够减少约20％的患者中观察到的高病毒负荷1,2。 EBV-CTL似乎也可以防止向EBV淋巴瘤发展，因为没有接受预防性CTL的患者出现这种恶性肿瘤，而对照组为11.5％。此外，接受CTL作为明显淋巴瘤治疗的六名患者中有五名可完成。在没有反应的患者中，肿瘤是通过删除了供体CTL系的两个CTL表位的病毒转化的，该病毒是特定于22的。 2.2与EBV特异性CTL的鼻咽癌免疫疗法的理由。 CTL疗法提供的主要优势是缺乏毒性。如果CTL有效地治疗复发性疾病，则可以用作鼻咽癌前线治疗的一部分，并允许降低放疗和化学疗法剂量，从而减少长期后遗症。由于已被证明是长期寿命和有效的，可在HSCT受体中预防和治疗与EBV相关的PTLD，因此它们有可能对具有其他EBV相关的恶性肿瘤患者有益，这些恶性肿瘤表达潜在的CTL靶标的病毒潜伏期蛋白。重要的是，NPC肿瘤细胞表达MHC I类分子以及肽转运蛋白TAP1和TAP2，因此能够在HLA I类MHC分子的背景下处理和呈现内源合成的蛋白质，以通过CTL23进行免疫识别。 3。淋巴稳态的破坏，以促进注入CTL的扩张和植入。 3.1 CTL输注之前T细胞耗竭的基本原理。在健康的成年人中，淋巴细胞数和子集成分保持在稳定的水平。由于感染，细胞丧失或其他影响引起的细胞数量的扰动通过知识率了解的稳态机制恢复为稳态水平。每个淋巴室或多或少地保持体内平衡，因此在缺乏T细胞的小鼠中，B细胞数保持与正常小鼠相同。同样，源自胸腺增殖并保持T细胞受体多样性的NANVE T细胞子群的调节独立于记忆T细胞调节，该记忆T细胞调节由抗原独立于抗原依赖性和抗原依赖性机制维持。在T细胞耗尽后，具有完全多样化的T细胞受体库的淋巴样重构取决于胸腺波素，但是无论淋巴隔室的大小如何，这都以稳定的速度持续。相反，记忆T细胞的膨胀是周围再现的主要来源25,26。 淋巴细胞数的调节可以通过计算机制或可用性“空间”来控制细胞因子或配体。尽管这些机制在免疫反应过程中被绕开，但脾脏，淋巴结和血液的大小增加，但是当抗原刺激呈落空时，稳定状态迅速恢复。在稳态情况下，注入的T细胞的扩展可能受到限制。然而，在T细胞不足的背景下，注入的细胞已被证明可以迅速增殖以恢复稳定的情况5。动物实验为我们提供了重要的规则，该规则在面临淋巴细胞减少症时调节体内平衡的恢复。首先，恢复赤字的T细胞增殖不取决于抗原刺激，尽管活化的T细胞具有增殖优势，在存在抗原24的情况下会增加。其次，一旦补充了周围池，就会减少进一步的扩张，从而防止注入淋巴细胞的无限膨胀。第三，即使使用少量细胞，也可以单次输注成熟的T淋巴细胞来快速重生。相反，在高剂量淋巴细胞输注的情况下，稳态是通过凋亡而不是增殖机制实现的。由于只能通过增殖来确定进入记忆池27，因此适当的细胞数的给药对于允许体内扩张至关重要，因此至关重要。最后，额外的知之甚少的机制保持周围多样性，并防止抗原驱动的寡克隆进展。在辐照淋巴细胞减少小鼠的多克隆自体T细胞中，当动物受到黑色素瘤或结肠癌细胞的挑战时，多克隆自体T细胞可防止肿瘤生长28。因此，通过增殖来稳态可以解释为什么在耗尽T细胞耗尽的BMT后，少量的多克隆EBV特异性CTL的输注，并在输液后长达八十个月持续：T细胞舱被耗尽了；注入的T细胞被激活。存在EBV抗原。相比之下，细胞凋亡的稳态可以解释为什么注入T细胞中的大量克隆T细胞在输注后迅速消耗的患者迅速消失，并且切勿进入记忆池29。 上述淋巴细胞稳态的机制表明，淋巴细胞耗竭后少数EBV特异性T细胞的施用可能允许体内显着膨胀，从而改善其抗肿瘤功能。此外，对抑制性CTL的功能的肿瘤浸润T细胞的耗竭可能是CTL输注之前淋巴细胞耗竭的另一种有利作用。在AIM 2的基本原理中进一步讨论了这种次要增益。淋巴样耗竭是为扩展采用转移的细胞扩大空间并促进其进入肿瘤部位的策略，已经显示出成功的证据。当黑色素瘤患者在大量高度激活的黑色素瘤特异性肿瘤浸润T细胞（TIL）之前接受环磷酰胺和氟达拉滨接受环磷酰胺和氟达滨时，观察到转移细胞的再生和增殖，以及转移性黑素瘤的回归。然而，尽管他们的扩张，一些患者仍然深刻地免疫功能低下，未能从这些注入的TIL中再生有效的免疫系统。这种不良的免疫重构造成的部分是由于所使用的细胞毒性药物对居民免疫系统的广泛和非特异性破坏所致。 T细胞耗竭的一种系统毒性较少的方法，尤其是对髓样系统，可能会使注入CTL的体内扩展并恢复残留的淋巴样室。靶向常见白细胞抗原的CD45 mAb将淋巴细胞室耗尽了约30天，而髓样系统在72小时内再生。结合其短期寿命（约8小时），允许在mAb给药后不久输注CTL，这些特征使CD45 mAb成为潜在安全有效的淋巴结凝集的方法。在C（7）节中讨论了我们在鼠和人类研究中对CD45 mAB的经验。 4。摘要和含义 我们假设，通过事先耗尽T细胞室，可以大大增加注入EBV+ NPC患者的EBV特异性CTL的抗肿瘤活性，从而允许扩大注入肿瘤特异性T细胞。我们认为，CD45 mAb会安全，实质上耗尽正常的T细胞，其短半衰期将允许在CD45 mAb给药后不久将EBV特异性CTL输注，从而在内源性T细胞恢复之前可以扩展。这项研究将提供有关MAB介导的T细胞耗竭能够增强采用转移的肿瘤特异性T细胞增殖的能力的重要信息。