Interleukin-12-responding asialoGM1+CD8+ central memory-type T cells as precursor cells for interferon-gamma-producing killer T cells

While investigating CD8(+) memory T cells in unimmunized C57BL/6 mice, we found that there were unique memory-type CD8(+) T cells expressing asialoGM1 (ASGM1), CD62L and CCR7 cell surface molecules, which occupied approximately 10% of CD8(+) T cells and 35% of CD44(+) memory CD8(+) T cells. Culture of freshly isolated ASGM1(+)CD8(+) T cells with interleukin (IL)-12 plus IL-2 caused the proliferation and generation of killer T cells. Moreover, ASGM1(+)CD8(+) T cells, but not ASGM1(-)CD8(+) T cells, produced high levels of interferon (IFN)-gamma in response to IL-12 plus IL-2. Although ASGM1(+)CD8(+) T cells showed no significant responses to IL-12 alone or IL-2 alone, pulse incubation of ASGM1(+)CD8(+) T cells with IL-12 at an earlier time (0-12 h), and subsequently with IL-2 at a later time (12-24 h), caused the same levels of proliferation, killer cell generation and IFN-gamma production as when they were incubated simultaneously with IL-12 plus IL-2 for 24 h. Thus, ASGM1(+)CD8(+) T cells appeared to respond to IL-12 directly to acquire IL-2 responsiveness and differentiate into IFN-gamma-producing killer T cells. Indeed, freshly isolated ASGM1(+)CD8(+) T cells, but not ASGM1(-)CD8(+) T cells, expressed higher levels of IL-12R beta2 mRNA. The fact that IL-12 administration in vivo caused the generation of ASGM1(+)CD8(+) killer T cells in an IFN-gamma-dependent manner further indicated a physiological significance of ASGM1(+)CD8(+) central memory-type T cells in IL-12-induced immunoregulation for the therapy of tumors and infectious diseases.     

脐血体外扩增干祖细胞和同时诱导分化T、NK和DCs免疫细胞

Umbilical cord blood stem cell transplantation (CB- SCT) has approached significant success in treatment of lethal congenital or malignant disorders, but CBSCT with low incidence of GVHD is associated with higher rates of delayed or failed engraftment and relapse than bone marrow transplants. This may be caused by immature im- mune cells, compared with the corresponding cells from adults. More studies have been reported that cord blood T lymphocytes are immature in phenotype and function…     

CD8+ T cell-mediated control of distant tumours following local photodynamic therapy is independent of CD4+ T cells and dependent on natural killer cells

Cancer survival rates decrease in the presence of disseminated disease. However, there are few therapies that are effective at eliminating the primary tumour while providing control of distant stage disease. Photodynamic therapy (PDT) is an FDA-approved modality that rapidly eliminates local tumours, resulting in cure of early disease and palliation of advanced disease. Numerous pre-clinical studies have shown that local PDT treatment of tumours enhances anti-tumour immunity. We hypothesised that enhancement of a systemic anti-tumour immune response might control the growth of tumours present outside the treatment field. To test this hypothesis we delivered PDT to subcutaneous (s.c.) tumours of mice bearing both s.c. and lung tumours and monitored the growth of the untreated lung tumours. Our results demonstrate that PDT of murine tumours provided durable inhibition of the growth of untreated lung tumours. The inhibition of the growth of tumours outside the treatment field was tumour-specific and dependent on the presence of CD8(+) T cells. This inhibition was accompanied by an increase in splenic anti-tumour cytolytic activity and by an increase in CD8(+) T cell infiltration into untreated tumours. Local PDT treatment led to enhanced anti-tumour immune memory that was evident 40 days after tumour treatment and was independent of CD4(+) T cells. CD8(+) T cell control of the growth of lung tumours present outside the treatment field following PDT was dependent upon the presence of natural killer (NK) cells. These results suggest that local PDT treatment of tumours lead to induction of an anti-tumour immune response capable of controlling the growth of tumours outside the treatment field and indicate that this modality has potential in the treatment of distant stage disease.     

Epstein-Barr virus nuclear antigen 1-specific CD4+ T cells directly kill Epstein-Barr virus-carrying natural killer and T cells

Epstein–Barr virus (EBV) nuclear antigen (EBNA)1 is expressed in every EBV-infected cell, regardless of the state of EBV infection. Although EBNA1 is thought to be a promising antigen for immunotherapy of all EBV-associated malignancies, it is less clear whether EBNA1-specific CD4⁺ T cells can act as direct effectors. Herein, we investigated the ability of CD4⁺ T-cell clones induced with overlapping peptides covering the C-terminal region of EBNA1, and identified minimal epitopes and their restricted major histocompatibility complex class II molecules. Of these, a novel epitope, EYHQEGGPD, was found to be presented by DRB1*0401, 0403 and 0406. Five CD4⁺ T-cell clones recognized endogenously processed and presented antigens on EBV-transformed lymphoblastoid cell lines (LCL) and one example proved capable of killing EBV-carrying natural killer (NK) and T-cell lines derived from patients with chronic active EBV infection (CAEBV). Identification of minimal epitopes facilitates design of peptide-based vaccines and our data suggest that EBNA1-specific CD4⁺ T cells may play roles as direct effectors for immunotherapy targeting EBV-carrying NK and T-cell malignancies. ( Cancer Sci 2008; 99: 1633–1642)     

Protection against MUC1 expressing mouse tumours by intra-muscular injection of MUC1 cDNA requires functional CD8+ and CD4+ T cells but does not require the MUC1 tandem repeat domain

Using a C57Bl/6 mouse model system, where intramuscular (i.m.) injection of full length (FL) MUC1 cDNA protects against subsequent challenge with MUC1-expressing syngeneic tumour cells, we have investigated the importance of the tandem repeat (TR) domain in the induction of T cell-dependent tumour rejection. A MUC1 construct engineered to remove the TR domain (MUC1 0TR) was found to be as effective as the full length MUC1 cDNA in inhibiting the growth of RMA MUC1 cells in C57Bl/6 mice. Protection by i.m. injection of either the FL-MUC1 cDNA or the MUC1 0TR construct depended on the presence of functional CD4+ and CD8+ T cells. Specific CD8+ T cell responses, however, could not be detected in vitro using mouse spleen cells taken after only cDNA injection, but only after challenge in vivo with MUC1-expressing tumour cells. To attempt to enhance the responses of CD4+ T cells, a cDNA construct was developed, where the extracellular domain of MUC1 was fused to the transmembrane and cytoplasmic domain of Lamp1 (MUC1/Lamp1). This construct was equally effective in inducing tumour rejection but did not induce MUC1-specific CTL in mice before challenge with MUC1-expressing tumour cells. Our results indicate that, in this model, T cell responses necessary for protection against MUC1-expressing tumours that are induced by IM injection of MUC1 cDNA are independent of the tandem repeat domain as well as the transmembrane and cytoplasmic domains. A low level of protection was seen with all constructs in BALB/c mice, which show a defect in Th1 responses. C57Bl/6xBALB/c hybrids were, however, well protected against both H2(d) and H2(b) expressing tumour challenge, emphasizing the importance of the host background.     

Natural killer receptors on CD8 T cells and natural killer cells from different HLA-C phenotypes in melanoma patients.

PURPOSE: Because immune mechanisms involved in cutaneous melanoma have not been fully elucidated, efforts have been made to achieve prognosis markers and potential targets for immune therapies, but they have not been entirely fruitful thus far. Therefore, the goal of this study was to investigate the involvement of early changes in CD8 T cells and CD56 natural killer (NK) cells expressing NK receptors in different HLA-C dimorphism groups of melanoma patients. EXPERIMENTAL DESIGN: CD8 T cells and CD56 NK cells were analyzed in 41 patients and 39 sex- and age-matched controls with different HLA-C genotypes by flow cytometry. HLA-C dimorphism at position 80 was tested by PCR sequence-specific primers and PCR sequence-specific oligonucleotide to examine whether it could mediate in the emergence of cells expressing killer cell immunoglobulin-like receptors. RESULTS: Thirty-five of 41 patients had benign sentinel node, and showed an imbalance in the absolute number of CD8(+)DR(+) or CD8(+)CD161(+) peripheral blood T cells according to the CD28 coexpression compared with controls. CD8(+)CD28(-)CD158a(+) T and CD56(+)CD158a(+) NK cells were significantly increased in HLA-C(Lys80) homozygous nonmetastatic patients, whereas only CD56(+)CD158a(+) NK cells increased in heterozygous ones. An up-regulation of the CD158a KIR receptor was also seen on NK cells but not in T cells of patients at advanced disease stages. CONCLUSIONS: This work provides, for the first time, evidence of immune activation in early stages of cutaneous melanoma, together with an increase of cells expressing CD158a in patients bearing the corresponding HLA-C ligand, which may be important to evaluate the disease progression and to use individualized immune therapeutic approaches.     

On the site and mode of antigen presentation for the initiation of clonal expansion of CD8 T cells specific for a natural tumor antigen

Because of the low frequency of antigen-specific T cells, early events in the activation of tumor-specific T cells in vivo have not been well characterized. There is still no direct documentation on where the clonal expansion begins and how tumor antigens are presented to the host CD8 T cells to initiate it. Here we used transgenic T cells specific for a natural tumor antigen P1A to evaluate the kinetics, location, and modes of antigen presentation for initiating CTL response in vivo. Our results demonstrate that the initial activation of P1A-specific T cells takes place in the lymphoid organs. The activated T cells then migrate into tumors, where they undergo accelerated division and acquire distinct activation markers. The site of initiation cannot be altered by either local expression of costimulatory molecules or by intratumor injection of na?ve T cells. Moreover, using genetic models that allow only one mode of antigen presentation, we show here that both cross-presentation of P1A by the host antigen-presenting cells, and direct antigen presentation and costimulation by the tumor cells are sufficient to initiate rapid T cell-clonal expansion in the lymphoid organ. These results provide direct evidence for two fundamental assumptions on the mechanisms of T-cell activation in vivo.     

Cutaneous CD56 positive natural killer and cytotoxic T-cell lymphomas

We report two cases of CD56 positive natural killer (NK) cell and cytotoxic T-cell cutaneous lymphomas and review the literature on these rare forms of non-Hodgkin's lymphoma. The first case was diagnosed to have extra nodal NK/T-cell lymphoma, nasal-type. She had a rapid downhill clinical course and died within 3 months of presentation. She had been started on systemic chemotherapy but did not respond. The second case was diagnosed as subcutaneous panniculitis-like T-cell lymphoma, CD56 positive variant. She presented with skin nodules that were quiescent for 10 years. Then the course of the disease suddenly changed and progressed rapidly. She had systemic chemotherapy and initially had a complete response, but she relapsed within 1 month of completion of chemotherapy. She then had partial response with further chemotherapy but relapsed rapidly. She died within 15 months of her lymphoma changing to its aggressive form. These cases illustrate the often poor prognosis of cutaneous CD56 positive lymphomas.     

CD3+CD56+CD8+ cells demonstrating a suppressor T cell-like function in the peripheral blood of colon cancer patients

We previously reported that HLA-unrestricted CTLs against MUC-1 were induced from colon cancer patients by stimulating peripheral blood lymphocytes (PBLs) with recombinant MUC-1 vaccinia virus (rVMUC-1). We have performed adoptive immunotherapy (AI) for two gastric and two colon cancer patients, using the rVMUC-1-stimulated T lymphocytes. A significant level of HLA-unrestricted cytotoxicity against MUC-1 was induced in the two colon cancer patients (pA and pB) during the first adoptive immunotherapy, but extremely reduced during the second AI. During the second stimulation phase, the rate of CD3+CD56+CD8+ cells were significantly increased and that of CD3+CD56-CD4+ cells were significantly decreased in the two colon cancer patients as compared to the first AI. CD3+CD56+CD8+ and CD3+CD56-CD4+ cells were isolated from the second AI of the colon cancer patient (pB) and designated as D856 and D4, respectively. The D4 cells demonstrated a high level of HLA-unrestricted CTL activity against MUC-1, but D856 cells did not. When D856 cells were mixed with D4 cells at a D856/D4 ratio of 1:3, 1:2, and 1:1 and used as effector cells, the HLA-unrestricted and MUC-1-specific CTL activity of D4 cells was suppressed in a D856/D4 ratio-dependent manner. Further, D856 cells were highly lytic for the D4 cells demonstrating HLA-unrestricted cytotoxicity against MUC-1. It is concluded that the reduction in HLA-unrestricted cytotoxicity against MUC-1 during the second AI is attributed to the D856 cells killing MUC-1-specific CTLs (D4). Thus, the CD3+CD56+ CD8+ cells seem likely to behave as a suppressor T cell.     

CD3+CD56+NKT细胞及CD3-CD56+NK细胞在恶性肿瘤患者的临床意义研究

背景与目的细胞毒性淋巴细胞在抗肿瘤免疫效应中发挥着重要作用,CD3 CD56 NKT细胞作为一类新的具有细胞毒性的效应细胞,目前关于其抗肿瘤意义的探讨主要集中于血液系统恶性疾病,而在实体肿瘤中的应用和临床价值研究尚少。本研究旨在初步探讨抗肿瘤细胞CD3 CD56 NKT细胞及CD3-CD56 NK细胞在恶性肿瘤患者外周血的表达状态及其临床意义。方法采用流式细胞术分析118例恶性肿瘤患者(55例肺癌患者和63例乳腺癌患者)及46例健康对照组外周血中的T细胞亚群及CD3 CD56 NKT细胞、CD3-CD56 NK细胞表达。结果恶性肿瘤患者组CD3 CD8 T细胞、CD3 CD56 NKT细胞以及CD3-CD56 NK细胞表达率均明显高于健康对照组(P<0.01)。肺癌患者中以CD3 CD8 T细胞和CD3 CD56 NKT细胞明显增加为主;乳腺癌患者中以CD3 CD56 NKT细胞和CD3-CD56 NK细胞明显增加为主。结论CD3 CD56 NKT细胞在肺癌和乳腺癌患者的抗肿瘤效应中占据重要地位,而CD3 CD8 CTL和CD3-CD56 NK细胞在不同类型肿瘤患者中具有不同的重要性。     

CD1d-mediated stimulation of natural killer T cells selectively activates hepatic natural killer cells to eliminate experimentally disseminated hepatoma cells in murine liver

Since hepatocellular carcinomas (HCCs) develop from transformed hepatocytes, sometimes in a multicentrical manner, immunological deletion of such small intrahepatic regions should be an important strategy to prevent HCC development. The liver contains abundant innate cell lineages including natural killer (NK) cells and natural killer T (NKT) cells, the latter of which become activated in a CD1d-restricted manner by alpha-galactosylceramide (alpha-GalCer). In our study, we investigated the anti-tumor effect elicited by alpha-GalCer administration against transplanted hepatoma cells in the liver, in comparison with that in extrahepatic sites. alpha-GalCer administration completely suppressed the growth of BNL 1MEA.7R.1 (BNL) hepatoma cells disseminated in the liver of syngeneic BALB/c mouse but had no anti-tumor effect on subcutaneously implanted BNL cells. Hepatic NKT cells became rapidly activated after alpha-GalCer administration compared to splenic NKT cells and then disappeared. Hepatic NK cells substantially increased their population as well as up-regulated their cytotoxic activity against BNL cells, but NK cells in other tissues, including the spleen, blood and lymph node, did not. Anti-asialo GM1 antibody treatment, which depleted NK cells in vivo, resulted in hepatic tumor formation in alpha-GalCer-treated mice, indicating the critical involvement of NK cells in the alpha-GalCer-induced anti-tumor effect in the liver. In conclusion, our study demonstrates clear differences in NK cell activation and anti-tumor effect through stimulation of NKT cells by alpha-GalCer between the liver and extrahepatic tissues. Sequential activation of these innate cell lineages may be an attractive strategy for controlling micro-disseminated hepatoma cells in the liver.     

选择性扩增人自然杀伤细胞的实验研究

背景与目的:自然杀伤(naturalkiller,NK)细胞具有很强的杀瘤活性,但体外扩增NK细胞的数量难以达到治疗肿瘤的目的。本实验探讨由人外周血单个核细胞(peripheralbloodmononuclearcells,PBMCs)和附壁Wilms肿瘤细胞株HFWT混合培养能否有效地诱导NK细胞。方法:采用PBMCs和HFWT细胞混合培养诱导扩增NK细胞,51Cr释放法和结晶紫染色法测定NK细胞的杀瘤活性;流式细胞计数仪检测培养细胞中CD3 、CD4 、CD8 、CD16 和CD56 细胞的比例。结果:HFWT对人NK细胞特别敏感,它比不表达MHC-Ⅰ类分子的K562细胞和其他附壁生长的肿瘤细胞株更有效地刺激PBMCs,从而选择性扩增NK细胞。健康人PBMCs在HFWT细胞中培养10～21天后,CD56 CD16 细胞占细胞总数50%以上。当效靶比为2∶1时,NK细胞杀伤80%的HFWT细胞,NK细胞的扩增需要HFWT细胞反复刺激PBMC。结论:从HFWT细胞株中可选择性扩增人NK细胞,将有助于开展临床肿瘤过继免疫疗法。     

人外周血CD56+NK细胞亚群表型和生物学特征

目的 探讨人CD56^＋NK细胞亚群、表型和生物学特征。方法 分离正常人外周血单个核细胞,利用多种抗体进行细胞表面和细胞内细胞毒效应分子（颗粒酶B和穿孔素）染色,通过流式细胞仪,在单个细胞水平上分析CD56^＋NK细胞亚群的表型及其生物学特征。结果 根据CD56分子表达密度的不同,将人外周血中NK细胞分为CD56^bright出和CD56^dim两大亚群。CD56^bright和CD56^dim细胞均表达CD95,CD56^bright细胞亚群中,CD95^bright和CD95^dim表达百分率分别为21．4％和77．0％;CD56^dim细胞亚群中,CD95^bright和CD95^dim表达百分率分别为23．1％和75．6％。与CD56^bright出细胞亚群相比,CD56^bright细胞表达CD8、颗粒酶B和穿孔素细胞的阳性率较高,分别为9．1％、6．2％和8．O％。在CD56^bright和CD56^bright亚群中,CD95^brightCD8^＋亚群高表达颗粒酶B和穿孔素。结论 人外周血CD56^＋NK细胞由不同表型和生物学特征的细胞亚群组成,CD56^dim”和CD56^dimCD8^＋NK细胞亚群在杀伤破坏靶细胞中发挥重要作用。     

Sustained antigen-specific antitumor recall response mediated by gene-modified CD4+ T helper-1 and CD8+ T cells

Given that specific subsets of T helper 1 (Th1) and T helper 2 (Th2) CD4(+) T cells have been shown to play key roles in tumor rejection models, we wanted to assess the contribution of either Th1 or Th2 CD4(+) cell subtypes for redirected T-cell immunotherapy. In this study, we have developed a novel method involving retroviral transduction and in vitro T-cell polarization to generate gene-engineered mouse CD4(+) Th1 and Th2 cells or T helper intermediate (Thi) cells expressing an anti-erbB2-CD28-zeta chimeric receptor. Gene-modified Th1 and Th2 polarized CD4(+) cells were characterized by the preferential secretion of IFN-gamma and interleukin-4, respectively, whereas Thi cells secreted both cytokines following receptor ligation. In adoptive transfer studies using an erbB2(+) lung metastasis model, complete survival of mice was observed when transduced Th1, Th2, or Thi CD4(+) cells were transferred in combination with an equivalent number of transduced CD8(+) T cells. Tumor rejection was consistently associated with transduced T cells at the tumor site and interleukin-2 secretion. However, the surviving mice treated with gene-modified Th1 CD4(+) cells were significantly more resistant to a subsequent challenge with a different erbB2(+) tumor (4T1.2) implanted s.c. This result correlated with both increased expansion of Th1 CD4(+) and CD8(+) T cells in the blood and a greater number of these cells localizing to the tumor site following rechallenge. These data support the use of gene-modified CD4(+) Th1 and CD8(+) T cells for mediating a sustained antitumor response.     

Regression of engineered myeloma cells secreting interferon-gamma-inducing factor is mediated by both CD4(+)/CD8(+) T and natural killer cells

IL-18 is a novel cytokine that stimulates T and NK cell activity and has potent antitumor effects. In this study, a mouse IL-18 gene was transfected into the mouse myeloma cell line J558. Our data demonstrated that (i) inoculation of 0.5x10(6) engineered tumor cells J558/IL-18 into syngeneic mice induced a Th1 dominant immune response and resulted in tumor regression in all 8/8 mice; (ii) the IL-18 antitumor effect was significantly decreased in mice depleted of either the CD4(+), or CD8(+), or NK cell subset, respectively but was completely abrogated in mice depleted of both CD4(+) and CD8(+) T cells; (iii) in vivo neutralization of IFN-gamma was accompanied by the growth of J558/IL-18 tumor in all the mice; and (iv) the J558/IL-18 tumor regression further induced protective immunity against a subsequent challenge by the parental J558 tumor, which is mediated by CD8(+) T cells as examined in the cytotoxicity assay in vitro and in the animal study in vivo. Taken together, our findings indicate that: (i) IL-18 can induce antitumor immune responses mediated by both CD4(+)/CD8(+) T cells and NK cells; and (ii) it is associated with IFN-gamma production. This study thus highlights the potential utility of IL-18 as an antitumor agent, a role that it can fulfil alone or in combination with other immunomodulatory cytokines such as IL-12.     

The in vivo expansion rate of properly stimulated transferred CD8+ T cells exceeds that of an aggressively growing mouse tumor

It has been hypothesized that rapidly dividing tumor cells can outpace adoptively transferred antitumor lymphocytes when tumors are large. However, this hypothesis is at odds with clinical observations indicating that bulky tumors can be destroyed by small numbers of adoptively transferred antitumor T cells. We sought to measure the relative growth rates of T cells and tumor cells in a model using transgenic CD8(+) T cells specific for the gp100(25-33) H-2D(b) epitope (called pmel-1) to treat large, well-established s.c. B16 melanoma. We tested the effect of the immunization using an altered peptide ligand vaccine alone or in combination with interleukin-2 (IL-2) by analyzing the kinetics of T-cell expansion using direct enumeration. We found that pmel-1 T cells proliferated explosively during a 5-day period following transfer. Calculations from net changes in population suggest that, at the peak of cell division, pmel-1 T cells divide at a rate of 5.3 hours per cell division, which was much faster than B16 tumor cells during optimal growth (24.9 hours per cell division). These results clearly indicate that the notion of a kinetic "race" between the tumor and the lymphocyte is no contest when adoptively transferred cells are stimulated with immunization and IL-2. When appropriately stimulated, tumor-reactive T-cell expansion can far exceed the growth of even an aggressively growing mouse tumor.     

Immune surveillance: both CD3+ CD4+ and CD3+ CD8+ T cells control in vivo growth of P815 mastocytoma.

The aim of this study was to examine whether a spontaneous immune response controls neoplastic growth in P815-bearing DBA/2 mice, and to characterize the cells involved in tumor resistance in vivo. Several cell lineages such as T-cell-receptor (TcR)-bearing T cells, NK cells and macrophages mediate some anti-tumor activity in vitro. P815 was chosen as a model because it is weakly immunogenic and is a good target both for tumor-specific, MHC-restricted CTL-mediated lysis and for MHC-unrestricted lysis exerted by long-term cultured lymphocytes or activated macrophages. Since most "NK-like activity" in freshly isolated populations appears to be associated with CD3- cells, whereas antigen-specific, MHC-restricted T cells mostly express CD3 determinants, CD3 was a good marker for evaluating the role of T cells and "NK" cells in tumor resistance in vivo. The survival of anti-CD3-treated animals that were inoculated with tumor cells was strongly reduced (mean survival time: 17 days vs. 40 days for the control group) and was associated with increased tumor growth rate. We followed the same approach to define the T-cell subset(s) that mediate(s) this immune response. Both CD4+ and CD8+ T cells were required for induction of immune control on neoplastic growth. The approach used has revealed the important role of CD4+ T cells in immune responses that control in vivo growth of a class-I-positive, class-II-negative tumor and suggests that these cells may play a central role in tumor resistance. Since CD4+ cells are activated by soluble, exogenous proteins, this finding may have important implications for immunotherapy.