Ex vivo expansion of tumor-infiltrating lymphocytes from nasopharyngeal carcinoma patients for adoptive immunotherapy

Establishing Epstein-Barr virus (EBV)-specific cytolytic T lymphocytes (EBV-CTLs) from peripheral blood mononuclear cells (PBMCs) for adoptive immunotherapy has been reported in EBV-associated malignancies including Hodgkin's lymphoma and nasopharyngeal carcinoma (NPC). In the current study,we performed ex vivo expansion of tumor-infiltrating lymphocytes (TILs) obtained from NPC biopsy specimens with a rapid expansion protocol using anti-CD3 monoclonal antibody (OKT3), recombinant human interleukin (IL)-2, and irradiated PBMCs from healthy donors to initiate the growth of TILs. Young TIL cultures comprised of more than 90% of CD3+T cells, a variable percentage of CD3+CD8+and CD3+CD4+T cells, and less than 10% of CD3-CD16+natural killer cells, a similar phenotype of EBV-CTL cultures from PBMCs. Interestingly, TIL cultures secreted high levels of the Th1 cytokines, interferon gamma (IFNγ) and tumor necrosis factor-alpha (TNF-α), and low levels of the Th2 cytokines, IL-4 and IL-10. Moreover, young TILs could recognize autologous EBV-transformed B lymphoblast cell lines, but not autologous EBV-negative blast cells or allogeneic EBV-negative tumor cells. Taken together, these data suggest that ex vivo expansion of TILs from NPC biopsy tissue is an appealing alternative method to establish T cell-based immunotherapy for NPC.

The emergence of tumor-infiltrating lymphocytes in nasopharyngeal carcinoma:Predictive value and immunotherapy implications

The clinical study of nasopharyngeal carcinoma(NPC)often reveals a large number of lymphocytes infiltrating the primary tumor site.As an important part of the tumor microenvironment,tumor-infiltrating lymphocytes(TILs)do not exist alone but as a complex multicellular population with high heterogeneity.TILs play an extremely significant role in the occurrence,development,invasion and metastasis of NPC.The latest research shows that they participate in tumorigenesis and treatment,and the composition,quantity,functional status and distribution of TILs subsets have good predictive value for the prognosis of NPC patients.TILs are an independent prognostic factor for TNM stage and significantly correlated with better prognosis.Additionally,adoptive immunotherapy using anti-tumor TILs has achieved good results in a variety of solid tumors including NPC.This review evaluates recent clinical and preclinical studies of NPC,summarizes the role of TILs in promoting and inhibiting tumor growth,evaluates the predictive value of TILs,and explores the potential benefits of TILs-based immunotherapy in the treatment of NPC.

KRAS-G12D mutation drives immune suppression and the primary resistance of anti-PD-1/PD-L1 immunotherapy in non-small cell lung cancer

Background:Although immune checkpoint inhibitors(ICIs)against programmed cell death protein 1(PD-1)and its ligand PD-L1 have demonstrated potency towards treating patients with non-small cell lung carcinoma(NSCLC),the potential association between Kirsten rat sarcoma viral oncogene homolog(KRAS)oncogene substitutions and the efficacy of ICIs remains unclear.In this study,we aimed to find point mutations in the KRAS gene resistant to ICIs and elucidate resistance mechanism.Methods:The association between KRAS variant status and the efficacy of ICIs was explored with a clinical cohort(n=74),and confirmed with a mouse model.In addition,the tumor immune microenvironment(TIME)of KRASmutant NSCLC,such as CD8+tumor-infiltrating lymphocytes(TILs)and PD-L1 level,was investigated.Cell lines expressing classic KRAS substitutions were used to explore signaling pathway activation involved in the formation of TIME.Furthermore,interventions that improved TIME were developed to increase responsiveness to ICIs.Results:We observed the inferior efficacy of ICIs in KRAS-G12D-mutant NSCLC.Based upon transcriptome data and immunostaining results from KRAS-mutant NSCLC,KRAS-G12D point mutation negatively correlated with PD-L1 level and secretion of chemokines CXCL10/CXCL11 that led to a decrease in CD8+TILs,which in turn yielded an immunosuppressive TIME.The analysis of cell lines overexpressing classic KRAS substitutions further revealed that KRAS-G12D mutation suppressed PD-L1 level via the P70S6K/PI3K/AKT axis and reduced CXCL10/CXCL11 levels by down-regulating high mobility group protein A2(HMGA2)level.Notably,paclitaxel,a chemotherapeutic agent,upregulated HMGA2 level,and in turn,stimulated the secretion of CXCL10/CXCL11.Moreover,PD-L1 blockade combined with paclitaxel significantly suppressed tumor growth compared with PD-L1 inhibitor monotherapy in a mouse model with KRAS-G12D-mutant lung adenocarcinoma.Further analyses revealed that the combined treatment significantly enhanced the recruitment of CD8+TILs via the up-regulation of CXCL10/CXCL11 levels.Results of clinical study also revealed the superior efficacy of chemo-immunotherapy in patients with KRAS-G12D-mutant NSCLC compared with ICI monotherapy.Conclusions:Our study elucidated the molecular mechanism by which KRASG12D mutation drives immunosuppression and enhances resistance of ICIs in NSCLC.Importantly,our findings demonstrate that ICIs in combination with chemotherapy may be more effective in patients with KRAS-G12D-mutant NSCLC.

LMPs特异性T淋巴细胞对鼻咽癌细胞的杀伤作用

目的:研究树突状细胞负载EB病毒潜伏膜蛋白(latent membrane proteins,LMPs)介导生成的LMPs特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)LMPs-CTL的生物学特性,观察其对LMPs阳性鼻咽癌细胞SUNE的杀伤作用。方法:用淋巴细胞分离液分离人外周血单核细胞,采用贴壁分离及白细胞介素(interleukin,IL)-4、粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)等细胞因子诱导成熟树突状细胞(dendritic cell,DC),通过DC细胞负载LMPs多肽抗原并递呈给同源T淋巴细胞,制备LMPs-CTL。CCK8法检测LMPs-CTL对SUNE细胞的杀伤作用;ELISA和羟基荧光素二醋酸盐琥珀酰亚胺脂(carboxy fluoroscein succinimidyl ester,CFSE)染色法检测LMPs-CTL分泌IFN-γ及其增殖。结果:LMPsCTL对SUNE细胞的杀伤率在12 h和24 h分别为(43.47±1.93)%和(77.15±3.18)%,显著高于对照组的(11.45±3.06)%和(24.27±13.20)%(P<0.05)。SUNE细胞刺激后,LMPs-CTL增殖能力较对照组有明显增强,IFN-γ分泌量达到(613.40±121.77)pg/ml,显著高于对照组(86.90±3.70)pg/ml(P<0.05)。结论:通过DC细胞负载LMPs混合多肽可诱导生成LMPsCTL,对LMPs阳性鼻咽癌细胞有较强的杀伤作用。

白细胞介素2行鼻咽癌局部免疫治疗的生物学效应和疗效观察

目的 :探讨既能充分发挥肿瘤浸润淋巴细胞的作用 ,又能降低费用 ,简单易行的对鼻咽癌进行免疫治疗的方法。方法 :将 6 0例 NPC病人随机分为两组 ,每组 30例 ,在放化疗前各行 IL- 2局部及全身治疗 ,分别观察其生物学效应和疗效。结果 :1局部组的原发灶有效率为 43.3% ,颈转移灶为 9.5 % ,全身组均无效。 2局部组治疗后原发灶内 T细胞数、嗜酸性细胞数及胶原纤维明显增多 (P <0 .0 5 ) ,有 7例患者原发灶周边出现大片条带状坏死灶 ,全身组无以上变化。 3局部组在用药时局部出现胀痛 ,5～ 10分钟后自行消退 ,有 1例出现低热。全身组在用药过程中 ,出现轻度血压下降及乏力各 15例 ,发热 12例 ,头昏 10例 ,均在治疗结束后自行消退。结论 :直接将小剂量 IL- 2注入瘤体或瘤周同样能激活、扩增 TIL 细胞而发挥其杀癌作用 ,其生物学效应和疗效显著 ,副作用小 ,简单易行 ,安全可靠。

广西地区三类人群EB病毒潜伏膜蛋白2特异性细胞免疫状态的研究

目的 初步探讨广西地区鼻咽癌患者、血清VCA/IgA阳性高危人群及血清VCA/IgA阴性健康人群EB病毒LMP2特异性细胞免疫状态的差异.方法 选择广西苍梧地区经血清学筛查VCA/IgA阳性高危人群志愿者60例,对照为血清VCA/IgA筛查阴性的健康志愿者20例,同时选取广西壮族自治区人民医院经病理确诊鼻咽癌患者134例,分离外周血单个核细胞（PBMC）,采用酶联免疫斑点法（ELISPOT）检测外周血中LMP2特异性细胞毒性T淋巴细胞（CTL）免疫水平.比较鼻咽癌患者与健康志愿者、高危人群外周血LMP2特异性CTL免疫状态的差异.结果 鼻咽癌患者LMP2特异性CTL免疫水平明显低于健康志愿者及血清EB病毒抗体阳性人群,差异有统计学意义（P＜0.01）;血清EB病毒抗体阳性三种不同滴度人群间LMP2特异性T细胞免疫反应差异有统计学意义（P=0.002）;鼻咽癌Ⅰ、Ⅱ期患者同Ⅲ期及Ⅳ期患者的LMP2特异性T细胞免疫反应存在差异（P＜0.01）.结论 三类人群间LMP2特异性CTL水平的差异可能是导致EB病毒长期潜伏感染并导致鼻咽癌发生的重要原因,针对LMP2的特异性免疫治疗有望降低鼻咽癌的发生率.