Assessment of the number of local cytotoxic T lymphocytes required for degradation of micrometer-size tumor spheroids

Adoptive immunotherapy with human cytotoxic T lymphocytes (CTL) is a promising cancer treatment. Previously we showed that human CTLs against various types of tumors can be efficiently produced by coculturing peripheral blood cells with target cells. The aims of this study were to simulate the interaction of CTLs and micrometer-size tumor tissues in vitro and to assess the required number of CTLs at local tumor sites for degradation of a tumor. Allogeneic CTLs against a human transitional cell carcinoma cell line and autologous CTLs against a renal cell carcinoma cell derived from a surgical specimen were generated. The cytotoxic activities of CTLs against tumor cells in monolayer culture and tumor spheroids formed in U-bottom 96-well culture plates were assessed. Both allogeneic and autologous CTLs showed greater destructive activity than lymphokine activated killer (LAK) cells against target tumor spheroids. CTLs inoculated at E/T ratios of 0.1 to 1 coexisted with the tumor spheroid for 5 to 6 days and then increased in number with apparently lethal activity against the tumor spheroid. In contrast to CTLs, the increase in LAK cell numbers was scarcely observed, and the proliferated LAK cells did not show cytotoxicity against the tumor spheroid. These observations suggest that, when a small number of CTLs reach a local tumor site, they can destroy micrometer-size tumors after considerable local proliferation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Recycled addition of CD4+ T cell-rich population for induction of human autologous cytotoxic T lymphocytes: A practically efficient method

When CD4⁺ T cell-rich population appears in theinitial trial in induction cultures of humanautologous cytotoxic T lymphocytes (CTL), the cultureresults frequently in no or weak killing activity andtherefore usually be discarded as an `unsuccessful'CTL induction culture. However, addition of theinitial CD4⁺ T cell-rich population enabledefficient induction of the autologous CTL in theensuing trials. The CTL thus generated exhibitedstronger killing activities against autologous braintumor cells and ovarian tumor cells than previouslyobserved. This simple recycling of the primed butinert CD4⁺ T cell-rich population for CTLinduction will promote clinical practice of adoptiveimmunotherapy of human tumors with autologous CTL.     

Induction of the Epstein-Barr virus latent membrane protein 2 antigen-specific cytotoxic T lymphocytes using human leukocyte antigen tetramer-based artificial antigen-presenting cells

Cytotoxic T lymphocytes (CTLs) specific for the Epstein-Barr virus (EBV) latent membraneprotein 2 (LMP2) antigen are important reagents for the treatment of some EBV-associated malignancies,such as EBV-positive Hodgkin's disease and nasopharyngeal carcinoma.However,the therapeutic amount ofCTLs is often hampered by the limited supply of antigen-presenting cells.To address this issue,an artificialantigen-presenting cell (aAPC) was made by coating a human leukocyte antigen (HLA)-pLMP2 tetramericcomplex,anti-CD28 antibody and CD54 molecule to a cell-sized latex bead,which provided the dual signalsrequired for T cell activation.By co-culture of the HLA-A2-LMP2 bearing aAPC and peripheral bloodmononuclear cells from HLA-A2 positive healthy donors,LMP2 antigen-specific CTLs were induced andexpanded in vitro.The specificity of the aAPC-induced CTLs was demonstrated by both HLA-A2-LMP2tetramer staining and cytotoxicity against HLA-A2-LMP2 bearing T2 cell,the cytotoxicity was inhibited bythe anti-HLA class Ⅰ antibody (W6/32).These results showed that LMP2 antigen-specific CTLs could beinduced and expanded in vitro by the HLA-A2-LMP2-bearing aAPC.Thus,aAPCs coated with an HLA-pLMP2 complex,anti-CD28 and CD54 might be promising tools for the enrichment of LMP2-specificCTLs for adoptive immunotherapy.     

A mathematical analysis of the interactions between immunogenic tumor cells and cytotoxic T lymphocytes.

Recent developments of biotechnology have enabled us to use immunotherapy against certain kinds of tumors in patients. However, it is reasonable to doubt if the immunotherapy can completely aid the rejection of tumors that have escaped from the immune system. In this paper, we propose a new mathematical model of tumor immunity by tumor-specific cytotoxic T lymphocytes (CTLs), since tumor-specific CTLs play an important role in tumor immunity. Using this model, we have mathematically investigated the interactions between immunogenic tumor cells (TCs) and tumor-specific CTLs and evaluated the availability of immunotherapies for tumors. The findings herein demonstrate that three kinds of dynamics of tumor immunity exist: i.e. (1) TCs continue to proliferate with CTLs; (2) TCs are rejected by CTLs; and (3) TCs equilibrate with CTLs, but with little possibility of the equilibrium. The findings also demonstrate that a sufficient increase in CTLs by immunotherapy can aid the rejection of TCs, but an insufficient increase in CTLs by immunotherapy causes only a transient regression of TCs. Clinically the findings mean that increasing tumor-specific CTLs, e.g., by vaccination or adoptive transfer of tumor-specific CTLs expanded ex vivo, can theoretically aid the rejection of TCs.     

Transfer of long-lasting tumor immunity by immune T cells from MHC congenic mice: Migration,survival and tumor-protectivity of cytotoxic donor cells

Immunocompetent B10.D2 (H-2^d) mice are able to reject the highly malignant lymphoma ESb of DBA/2 (H-2^d) origin very effectively. Seven days after intravenous injection of the ESb tumor cells, B10.D2 mice developed a strong tumor-rejection response which was associated with the generation of anti-tumor T cells in their spleens with direct cytotoxic activity. Most of the cytotoxic potential was directed against the minor histocompatibility differences as demonstrated by the lysis of unrelated DBA/2 derived Eb tumor cells and normal DBA/2 but no B10.D2 derived ConA lymphoblasts. A previously performed clonal analysis, however, revealed a minority population of CTL clones which specifically recognized the ESb specific transplantation antigen (ESb-TATA). When transferred systemically into DBA/2 mice, the B10.D2 anti-ESb immune spleen cells could delay the outgrowth of s.c. transplanted ESb tumor cells. When the ESb tumor cells were experimentally distributed in a s.c. implanted sponge-matrix, the i.v. injected B10.D2 immune cells could confer complete protective immunity against the metastatic tumor, provided the recipients were pre-treated with 5 Gy to allow a better take of the allogeneic cells. The distribution of intravenously injected B 10D2 donor spleen cells was assessed in the recipients up to 50 days by cytotoxicity testing and assaying for the expression of the ₂ microglobulin allelic form b ( ₂m^b). These tests revealed a high propensity of donor cells to populate the spleen and lymph nodes of the DBA/2 recipients. Again this was particularly marked in sublethally irradiated mice where a long-lasting lymphoid chimerism was established.     

Identification of melanoma-specific peptide epitopes by HLA-A2.1-restricted cytotoxic T lymphocytes

HLA-A2.1-associated peptides, extracted from human melanoma cells, were used to study epitopes for melanoma-specific HLA-A2.1-restricted cytotoxic T lymphocytes （CTLs） by epitope reconstitution, active peptide sequence characterization and synthetic peptide verification. CTL were generated from tumor-involved nodes by in vitro stimulation, initially with autologous melanoma cells and subsequently with allogeneic HLA-A2.1 positive melanoma cells. The CTLs could lyse autologous and aUogeneic HLA-A2. 1 positive melanomas, but not HLA-A2.1 negative melanomas or HLA-A2.1 positive non-melanomas. The lysis of melanomas could be inhibited by anti-CD3, anti-HLA class I and anti-HLA-A2.1 monoclonal antibodies. HLA-A2.1 molecules were purified from detergent-solubilized human melanoma cells by immunoaffinity column chromatography and further fractionated by reversed phase high performance liquid chromatography. The fractions were assessed for their ability to reconstitute melanoma-specific epitopes with HLA-A2.1 positive antigen-processing mutant T2 cells. Three reconstitution peaks were observed in lactate dehydrogenase release assay. Mass spectrometry and ion-exchange high performance liquid chromatography analysis were used to identify peptide epitopes. Peptides with a mass-to-charge ratio of 948 usually consist of nine amino acid residues. The data from reconstitution experiments confirmed that the synthetic peptides contained epitopes and that the peptides associated with HLA-A2.1 and recognized by melanoma-specific CTL were present in these different melanoma cells. These peptides could be potentially exploited in novel peptide-based antitumor vaccines in immunotherapy for CTL.     

In vitro induction of HLA-A2402-restricted and carcinoembryonic-antigen-specific cytotoxic T lymphocytes on fixed autologous peripheral blood cells

HLA-A2402-restricted and carcinoembryonic-antigen(CEA)-specific cytotoxic T lymphocytes (CTL) were induced by culturing human peripheral blood mononuclear cells (PBMC) on formalin-fixed autologous adhesive PBMC that had been loaded with CEA-bound latex beads. The CTL killed the CEA-producing HLA-type matched cancer cells, but not the non-producers of CEA, at an effector/target ratio of 10 within 24 h. On the basis of available HLA-A24-binding peptides, we have also attempted to identify the epitope peptide recognized by the CTL. The peptide CEA652(9), TYACFVSNL, stimulated the CTL most strongly when pulsed on HLA-A2402-expressing target cells. The other nine peptides so far tested were also active, but less efficient in their effect on CTL. The CTL failed to kill target cells pulsed with the HLA-A2-binding CEA peptide, CAP-1. The CTL were also generated on the fixed adherent cells previously pulsed with the peptide CEA652(9). Cytotoxic activity of the CTL was inhibited by monoclonal antibodies against CD3, CD8, and MHC class I molecules. These results suggest that human autologous CTL will be inducible on the autologous fixed PBMC without use of the cultured target cancer cells if tumor antigenic protein is available. Received: 31 December 1997 / Accepted: 4 May 1998     

Long-term serum/plasma-free culture of human cytotoxic T lymphocytes induced from peripheral blood mononuclear cells

Tumor-specific human cytotoxic T lymphocytes (CTL) were induced by co-culturing peripheral blood mononuclear cells with X-ray-irradiated human lung squamous carcinoma cells, SQ-5, in the medium supplemented with interleukin(IL)-1, IL-2, IL-4 and IL-6, and 5% autologous plasma for 3 or 5 days. The CTL grew in serum/plasma-free medium containing these four interleukins and 0.5% bovine serum albumin for over a month and maintained kiling activity of target cells within 48 h at an effector/target ratio of 1.25. Their growth was essentially dependent on the target SQ-5 cells, which were renewed every 5 days. Under these conditions, IL-4 and IL-6 could be omitted. When anti-CD3 monoclonal antibody was added to the serum/plasma-free medium supplemented with IL-1 and IL-2, the target tumor cells were not required to maintain the specific killing activity of the CTL. A large number of CTL (10¹¹) were obtained in 35 days.     

Anti-CD20scFv/IgGFc/CD80/CD28/zeta转染人T淋巴细胞对原代CLL细胞的作用

目的:将已成功构建表达anti-CD20scFv/CD80/CD28/zeta转染人T淋巴细胞,体外观察该类细胞特异性清除CD20+原代慢性淋巴细胞白血病(CLL)细胞的能力,为肿瘤的过继免疫治疗提供新思路。方法:将本室成功构建的含anti-CD20scFv/IgGFc/CD80片段的PLNCX质粒,转染PA317包装细胞,挑取高滴度的包装细胞株收获逆转录病毒,用收获的病毒感染刺激分裂的人外周血T细胞,经G418筛选后与CD20+原代CLL细胞在体外共同培养,在显微镜下观察CD20+的原代CLL细胞生长状态,ELISA检测试剂盒检测T细胞分泌细胞因子的功能。结果:重组基因修饰的T细胞能在体外杀伤CD20+原代CLL细胞,而对CD20-细胞无杀伤作用;同时靶细胞为CD20+组上清液中IL-2(1301.00pg/ml)和IFN-γ(602.18pg/ml)水平与CD20-组相比明显升高。结论:嵌合锚定T细胞能够成功构建;该类T细胞在体外能特异性杀伤CD20+的原代CLL细胞。     

Comparison of cytotoxic T lymphocyte responses against pancreatic cancer induced by dendritic cells transfected with total tumor RNA and fusion hybrided with tumor cell

Pancreatic cancer (PC) is a deadly human malignancy. Dendritic cell (DC)-based immunotherapy with whole tumor antigens demonstrates potential efficiency in cancer treatment. Tumor RNA and tumor fusion hybrid cells are sources of whole tumor antigens for preparing DC tumor vaccines. However, the efficacy of these sources in eliciting immune responses against PC has not yet to be directly compared. In the present study, patient-derived PC cells and DCs were fused (DC–tumor hybrids) and primary cultured PC cell-derived total RNA was electroporated into autologous DCs (DC–tumor RNA). The antitumor immune responses induced by DC–tumor hybrids and DC–tumor RNA were compared directly. The results showed that both RNA and hybrid methodologies could induce tumor-specific cytotoxic T lymphocyte (CTL) responses, but pulsing DCs with total tumor RNA could induce a higher frequency of activated CTLs and T-helper cells than fusing DCs with autologous tumor cells. In addition, DC–tumor RNA triggered stronger autologous tumor cell lysis than DC–tumor hybrids. It could be concluded that DCs pulsed with whole tumor RNA are superior to those fused with tumor cells in priming anti-PC CTL responses. Electroporation with total tumor RNA may be more suitable for DC-based PC vaccination.     

HBsAg-redirected T cells exhibit antiviral activity in HBV-infected human liver chimeric mice

Background

Chronic hepatitis B virus (HBV) infection remains incurable. Although HBsAg-specific chimeric antigen receptor (HBsAg-CAR) T cells have been generated, they have not been tested in animal models with authentic HBV infection.

Rab27a is required for regulated secretion in cytotoxic T lymphocytes

Rab27a activity is affected in several mouse models of human disease including Griscelli (ashen mice) and Hermansky-Pudlak (gunmetal mice) syndromes. A loss of function mutation occurs in the Rab27a gene in ashen (ash), whereas in gunmetal (gm) Rab27a dysfunction is secondary to a mutation in the alpha subunit of Rab geranylgeranyl transferase, an enzyme required for prenylation and activation of Rabs. We show here that Rab27a is normally expressed in cytotoxic T lymphocytes (CTLs), but absent in ashen homozygotes (ash/ash). Cytotoxicity and secretion assays show that ash/ash CTLs are unable to kill target cells or to secrete granzyme A and hexosaminidase. By immunofluorescence and electron microscopy, we show polarization but no membrane docking of ash/ash lytic granules at the immunological synapse. In gunmetal CTLs, we show underprenylation and redistribution of Rab27a to the cytosol, implying reduced activity. Gunmetal CTLs show a reduced ability to kill target cells but retain the ability to secrete hexosaminidase and granzyme A. However, only some of the granules polarize to the immunological synapse, and many remain dispersed around the periphery of the CTLs. These results demonstrate that Rab27a is required in a final secretory step and that other Rab proteins also affected in gunmetal are likely to be involved in polarization of the granules to the immunological synapse.     

Cellular interaction against autologous tumor cells between IL-2-cultured lymphocytes and fresh peripheral blood lymphocytes in patients with breast cancer given immuno-chemotherapy

In patients with Stage II or III breast cancer and in patients with liver metastases from breast cancer, we examined cellular interaction in the cytotoxicity against autologous tumor cells by interleukin-2(IL-2)-cultured lymphocytes (CL) and fresh peripheral blood lymphocytes (FPBL) treated with immunochemotherapy including OK-432 and cyclophosphamide. In flow cytometric analysis, CD8 + CD11b+ and CD16+ cells significantly decreased after immuno-chemotherapy in both groups of patients. A protocol study in Stage II or III breast cancer patients showed suppressive activity of FPBL on the cytotoxic activity of CL in 3/9 of the non-treatment group but no suppressive activity and enhancing activity in 3/7 in the immuno-chemotherapy group. Moreover, in 19 patients with liver metastases from breast cancer treated with immuno-chemotherapy including adoptive immunotherapy, FPBL in 6/19 showed enhancing activity, and in 8/19 suppressive activity in the lysis of autologous tumor cells. In assaysin vitro using autologous and allogeneic tumor cells, FPBL showed a partial specificity in cellular interaction against autologous tumor cells. CD4-depleted FPBL inhibited cytotoxicity of CL, while CD8-depleted FPBL enhanced cytotoxicity of CL in patients with liver metastases. These results suggest that immuno-chemotherapy eliminates the suppressive population in FPBL and may induce tumor regression if combined with adoptive immunotherapy using CL.Abbreviations IL-2 interleukin-2 - CL IL-2-cultured lymphocytes - FPBL fresh peripheral blood lymphocytes - AIT adoptive immunotherapy     

OK-432 and IL-2-augmental cytotoxicity of human natural killer cells and cytotoxic T lymphocytes at the clonal level

Abstract The biology response modifiers OK-432 and interleukin-2 (IL-2) were found to enhance the lytic capacity of cloned CD3⁻ natural killer (NK) cells and CD3⁺ T cells. With respect to NK cells, only those clones with a high proliferative capacity and cultured without phytohaemagglutinin (PHA) responded with enhaced lytic capacity to OK-432. OK-432, but not IL-2, was found to augment the antibody-dependent cellular cytotoxicity of cloned NK cells. With T-cell clones, OK-432 augmented the cellular cytotoxicity of CD3⁺8⁺ but not that of CD3⁺4⁺ cytotoxic T lymphocytes, while IL-2 augmented the cytotoxicity of both types of clone. Taken together, these results indicate that OK-432-augmented lytic capacity is not restricted to NK cells and its pathway of action may be independent of IL-2.     

Multicenter phase 1/2 application of adenovirus-specific T cells in high-risk pediatric patients after allogeneic stem cell transplantation

Background

Adenovirus (ADV) reactivation can cause significant morbidity and mortality in children after allogeneic stem cell transplantation. Antiviral drugs can control viremia, but viral clearance requires recovery of cell-mediated immunity.

Chinese medicine,Coix seeds increase peripheral cytotoxic T and NK cells

Coix seeds, a Chinese medicine have been used in Japan and reported to be effective in patients with verruca vulgaris and verrucae planae juveniles. We investigated thein vivo effects on lymphocyte subsets in seven healthy volunteers who took six tablets of Coix seeds three times a day (a typical dose) for four weeks. Leukocyte counts and the percentage of total lymphocytes did not change but the percentages of CD3^–CD56⁺ cells and CD16⁺CD57^– cells increased significantly. These results indicate that Coix seeds increase peripheral cytotoxic lymphocytes and may be effective to viral infection through the enhancement of cytotoxic activity.     

Cross-primed CD8+ cytotoxic T cells induce severe Helicobacter-associated gastritis in the absence of CD4+ T cells

BACKGROUND: Although previous studies have reported important roles of CD4(+) type 1-helper T cells and regulatory T cells in Helicobacter-associated gastritis, the significance of CD8(+) cytotoxic T cells remains unknown. To study the roles of CD8(+) T cells, we examined the immune response in the gastric mucosa of Helicobacter felis-infected major histocompatibility complex (MHC) class II-deficient (II(-/-)) mice, which lack CD4(+) T cells. MATERIALS AND METHODS: Stomachs from H. felis-infected wild-type and infected MHC II(-/-) mice were examined histologically and immunohistochemically. Gastric acidity and serum levels of anti-H. felis antibodies were measured. The expression of pro-inflammatory and anti-inflammatory cytokine, Fas-ligand, perforin, and Foxp3 genes in the gastric mucosa was investigated. RESULTS: H. felis-infected MHC II(-/-) mice developed severe gastritis, accompanied by marked infiltration of CD8(+) cells. At 1 and 2 months after inoculation, mucosal inflammation and atrophy were more severe in MHC II(-/-) mice, although gastritis had reached similar advanced stages at 3 months after inoculation. There was little infiltration of CD4(+) cells, and no Foxp3-positive cells were detected in the gastric mucosa of the infected MHC II(-/-) mice. The expression of the interleukin-1beta and Fas-ligand genes was up regulated, but that of Foxp3 was down regulated in the infected MHC II(-/-) mice. Serum levels of anti-H. felis antibodies were lower in the infected MHC II(-/-) mice, despite severe gastritis. CONCLUSIONS: The present study suggests that cross-primed CD8(+) cytotoxic T cells can induce severe H.-associated gastritis in the absence of CD4(+) helper T cells and that Foxp3-positive cells may have an important role in the control of gastric inflammation.     

Induction of cytotoxic T lymphocytes specific to malignant glioma using T2 cells pulsed with HLA-A2-restricted interleukin-13 receptor alpha 2 peptide in vitro

Interleukin-13 receptor α2 （IL-13Rα2） is a glioma-restricted cell-surface epitope not otherwise detected within the central nervous system. The present study is a report of a novel approach of targeting malignant glioma with IL-1 3Rα2-specific cytotoxic T lymphocyte （CTL） induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with human leukocyte antigen （HLA）-A2-restricted IL-1 3Rα2345-353 peptide-pulsed T2 cells. The induced CTL showed specific lysis against T2 cells pulsed with the peptide and HLA-A2^＋ glioma cells expressing IL- 1 3Rα2345-353, while HLA-A2 glioma cell lines that express IL-13Rα2345-353 could not be recognized by CTL. The peptide-specific activity was inhibited by anti-HLA class I monoclonal antibody. These results suggest that the induced CTL specific for IL-1 3Rα2345-353 peptide could be a potential target of specific immunotherapy for HLA-A2 patients with malignant glioma.     

Activation of cytotoxic T lymphocytes against CML28-bearing tumors by dendritic cells transduced with a recombinant adeno-associated virus encoding the CML28 gene

Induction of anti-tumor immune responses by dendritic cells (DCs) transduced with a recombinant adeno-associated virus type 2 (rAAV2) encoding tumor antigens is considered a promising approach for cancer vaccine development. CML28, a novel antigen with the properties of cancer/testis (CT) antigens, is an attractive target for antigen-specific immunotherapy. Here we investigated the feasibility of inducing CML28-specific cytotoxic T lymphocyte (CTL) responses using DCs transduced with the rAAV2 vectors containing the CML28 gene (rAAV/CML28). Using an adenovirus-free packaging system, rAAV/CML28 was generated. The transduction efficiency of rAAV/CML28 in DCs increased in a multiplicity of infection (MOI)-dependent manner. The rAAV/CML28 transduction did not impair DC maturation, but even enhanced the CD80 expression. The rAAV/CML28-transduced DCs induced CML28-specific CTLs which exhibited a MHC class I-mediated antigen-specific lytic activity against CML28-bearing tumor cell lines (HepG2 and MCF-7) as well as the primary leukemia blasts. These findings suggest that rAAV/CML28-transduced DCs vaccine may serve as a feasible approach for the treatment of CML28-associated cancers.     

Missing HLA class I expression on Daudi cells unveils cytotoxic and proliferative responses of human gammadelta T lymphocytes

The major subset of human blood gammadelta T lymphocytes expresses the variable-region genes Vgamma9 and Vdelta2. These cells recognize non-peptidic phosphoantigens that are present in some microbial extracts, as well as the beta(2)-microglobulin-deficient Burkitt's lymphoma Daudi. Most cytotoxic human Vgamma9/Vdelta2 T cells express inhibitory natural killer cell receptors for HLA class I that downmodulate the responses of the gammadelta T lymphocytes against HLA class I expressing cells. In this study we show that transfection of the human beta(2)-microglobulin cDNA into Daudi cells markedly inhibits the cytotoxic and proliferative responses of human Vgamma9/Vdelta2 T cells. This provides direct evidence that the "innate" specificity of human Vgamma9/Vdelta2 T-lymphocytes for Daudi cells is uncovered by the loss of beta(2)m by Daudi. However, Daudi cells that express HLA class I in association with mouse beta(2)m at the cell surface are recognized by human Vgamma9/Vdelta2 T cells close to the same degree as the parental HLA class I deficient Daudi cell line. Thus, proper conformation of the HLA class I molecules is required for binding to natural killer cell receptors. Cloning of the HLA class I A, B, and C molecules of Daudi cells and transfer of the individual HLA class I molecules of Daudi cells into the HLA class I deficient recipient cell lines.221 and C1R demonstrate that for some human gammadelta T-cell clones cytolysis can be entirely inhibited by single HLA class I alleles while for other clones single HLA class I alleles only partially inhibit cytotoxicity. Thus, most human Vgamma9/Vdelta2 T cells represent a population of killer cells that evolved like NK cells to destroy target cells that have lost expression of individual HLA class I molecules but with a specificity that is determined by the Vgamma9/Vdelta2 TCR.