Characterization of a chimeric T-cell receptor with specificity for the Hodgkin's lymphoma-associated CD30 antigen

Recombinant receptors with antibody-like specificity for tumor-associated antigens were shown to direct specifically T cells to target tumor cells. Hodgkin and Reed-Sternberg cells, the malignant cell population in Hodgkin's lymphoma, express high amounts of the cell surface antigen CD30. An anti-CD30 T-cell receptor with cellular activation properties is expected to graft T cells with specificity to Hodgkin cells. Here, the authors characterize a chimeric T-cell receptor with an extracellular domain consisting of the single-chain antibody fragment HRS3-scFv with specificity for the CD30 antigen and intracellular domain of the signal transducing part of the Fc-epsilon-I-gamma receptor. The HRS3-scFv was derived from the monoclonal anti-CD30 antibody HRS3 and retained specificity for the CD30 antigen. The recombinant HRS3-scFv-gamma receptor was expressed under control of the RSV-LTR after transfection into MD45 T-cells. The chimeric receptor protein is detected and analyzed by enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation. Expression of the chimeric receptor converts MD45 T cells to specificity for CD30+ lymphoma cells. Specific cross-linking of the chimeric receptor with antigen resulted in cytolytic reactivity against CD30+ tumor cells in vitro. The results demonstrate that the chimeric receptor HRS3-scFv-gamma converts T cells to a specific MHC-unrestricted cytolytic response against CD30+ tumor cells offering an alternative strategy in cellular immunotherapy of Hodgkin's disease.     

Targeting of natural killer-like T immunologic effector cells against leukemia and lymphoma cells by reverse antibody-dependent cellular cytotoxicity

Recently, highly efficient natural killer-like T immunologic effector cells called cytokine-induced killer (CIK) cells have been described. Most interestingly, CIK cells have been shown to eradicate established human lymphoma cells in a severe combined immunodeficient (SCID) mouse xenograft model in vivo. The current study was aimed at increasing the sensitivity of leukemia and lymphoma cells to CIK cells. In particular, the authors wanted to target CIK cells to leukemia and lymphoma cells via reverse antibody-dependent cellular cytotoxicity. Binding of an anti-CD3 monoclonal antibody to CIK cell cultures derived from patients with lymphoma was shown using flow cytometric analysis. For the target side, several B-cell lines were found to express CD19 on the cell surface. There was an impressive increase in sensitivity to CIK-mediated lysis of various lymphoma and leukemia cell lines by preincubation of the targets with a monoclonal antibody against CD3. This increase could be partially blocked by preincubation with anti-CD16 (Fc receptor III) and anti-CD32 (Fc receptor II) antibodies. These data suggest that the increase in cytotoxic activity is caused by Fc receptor-mediated antibody binding. Cytotoxic activity could be further increased by adding an anti-CD28 antibody in addition to anti-CD3. Finally, there was a further increase in sensitivity to CIK-mediated lysis of CD19+ malignant cells using the bispecific OKT3xHD37 antibody with specificity against CD3 and CD19. Interestingly, preincubation of malignant cells with an anti-CD3 monoclonal antibody followed by addition of the bispecific OKT3xHD37 antibody led to a further increase of cytotoxic sensitivity compared with the addition of the bispecific antibody alone. In conclusion, these data suggest that cytotoxic activity of immunologic effector cells can be increased not only by using the bispecific antibody OKT3xHD37 in vitro but also by preincubation of CD19+ leukemia and lymphoma cells with a monoclonal antibody against CD3. In addition, the immunostimulatory effect of the bispecific antibody OKT3xHD37 can be further increased by adding a monoclonal antibody against CD3.     

Autologous lymphoma vaccines induce human T cell responses against multiple, unique epitopes

The clonotypic surface Ig receptor expressed by malignant B cells, idiotype, is a tumor-specific antigen and an attractive target for active immunotherapy. While Ab's specific for tumor idiotype have been well described in patients with B cell malignancies, the precise antigenic epitopes in human idiotype recognized by autologous T cells remain largely unknown. We report here that T cell lines generated from lymphoma patients actively immunized with idiotype protein specifically recognized multiple, unique immunodominant epitopes in autologous tumor idiotype. Synthetic peptides corresponding to hypervariable, but not framework, regions of Ig heavy chain specifically stimulated CD4(+) and CD8(+) T cells to proliferate and secrete proinflammatory cytokines in an MHC-associated manner. Detailed analysis revealed a minimal determinant of an immunodominant epitope, comprising critical residues at the amino terminus that may be a product of somatic hypermutation. Association of idiotype-specific T cell responses with previously documented molecular remissions in idiotype-vaccinated patients suggests that the newly identified T cell epitopes may be clinically relevant. Such antigenic epitopes may serve as candidates for novel peptide-vaccine strategies, and as tools to selectively expand tumor antigen-specific T cells for adoptive immunotherapy and for monitoring T cell immunity in vaccinated patients.     

Cross-priming of naive CD8 T cells against melanoma antigens using dendritic cells loaded with killed allogeneic melanoma cells

The goal of tumor immunotherapy is to elicit immune responses against autologous tumors. It would be highly desirable that such responses include multiple T cell clones against multiple tumor antigens. This could be obtained using the antigen presenting capacity of dendritic cells (DCs) and cross-priming. That is, one could load the DC with tumor lines of any human histocompatibility leukocyte antigen (HLA) type to elicit T cell responses against the autologous tumor. In this study, we show that human DCs derived from monocytes and loaded with killed melanoma cells prime naive CD45RA(+)CD27(+)CD8(+) T cells against the four shared melanoma antigens: MAGE-3, gp100, tyrosinase, and MART-1. HLA-A201(+) naive T cells primed by DCs loaded with HLA-A201(-) melanoma cells are able to kill several HLA-A201(+) melanoma targets. Cytotoxic T lymphocyte priming towards melanoma antigens is also obtained with cells from metastatic melanoma patients. This demonstration of cross-priming against shared tumor antigens builds the basis for using allogeneic tumor cell lines to deliver tumor antigens to DCs for vaccination protocols.     

Generation of an HLA-restricted cytotoxic T cell line reactive against cultured tumor cells from a patient infected with human T cell leukemia/lymphoma virus

Lymphocytes from a patient who had an unusually long survival after therapy for a human T cell leukemia/lymphoma virus (HTLV)-associated T cell lymphoma were stimulated in vitro with an autologous tumor cell line, and the generation of cytotoxic T lymphocytes (CTL) was studied. CTL generated were directed against autologous (HTLV-associated tumor cells. These propagated CTL were OKT3+, OKT4-, and OKT8+. The cytotoxic activity required target tumor cells that were infected with HTLV and also expressed histocompatibility antigens in common with the patient, suggesting a major histocompatibility complex-restricted associative recognition of target antigens expressed on the tumor cell membrane.     

Melanoma cells present a MAGE-3 epitope to CD4(+) cytotoxic T cells in association with histocompatibility leukocyte antigen DR11

In this study we used TEPITOPE, a new epitope prediction software, to identify sequence segments on the MAGE-3 protein with promiscuous binding to histocompatibility leukocyte antigen (HLA)-DR molecules. Synthetic peptides corresponding to the identified sequences were synthesized and used to propagate CD4(+) T cells from the blood of a healthy donor. CD4(+) T cells strongly recognized MAGE-3281-295 and, to a lesser extent, MAGE-3141-155 and MAGE-3146-160. Moreover, CD4(+) T cells proliferated in the presence of recombinant MAGE-3 after processing and presentation by autologous antigen presenting cells, demonstrating that the MAGE-3 epitopes recognized are naturally processed. CD4(+) T cells, mostly of the T helper 1 type, showed specific lytic activity against HLA-DR11/MAGE-3-positive melanoma cells. Cold target inhibition experiments demonstrated indeed that the CD4(+) T cells recognized MAGE-3281-295 in association with HLA-DR11 on melanoma cells. This is the first evidence that a tumor-specific shared antigen forms CD4(+) T cell epitopes. Furthermore, we validated the use of algorithms for the prediction of promiscuous CD4(+) T cell epitopes, thus opening the possibility of wide application to other tumor-associated antigens. These results have direct implications for cancer immunotherapy in the design of peptide-based vaccines with tumor-specific CD4(+) T cell epitopes.     

抗CD20scFv/CD80/CD28/ζ重组基因修饰T细胞的构建与靶向杀伤效果

目的构建表达抗CD20scFv／CD80／CD28／ζ重组基因修饰T细胞，体外观察其特异性清除CD20^＋淋巴瘤细胞的能力，为肿瘤的过继免疫治疗提供新思路。方法采用DNA重组技术将pBULLET上的CD28-ζcDNA插入到已含有抗CD20scFv／CD80的真核表达载体pLNCX质粒上，转染PA317包装细胞，挑取高滴度的包装细胞收获病毒，用收获的病毒感染刺激分裂的人外周血T淋巴细胞，经G418筛选1周后与CD20^＋淋巴瘤细胞株Daudi、Raji在体外共同培养，用非放射性的细胞杀伤试剂盒和ELISA检测试剂盒分别检测T细胞的抗原特异性杀伤和细胞因子分泌的功能。结果酶切及测序鉴定均证实pLNCX／抗CD20seFv／CD80／CD28／ζ构建成功，重组基因修饰的T细胞能在体外杀伤CD20^＋淋巴瘤细胞株Daudi、Raji，而对CD20^＋细胞株K562无杀伤作用，同时CD20^＋靶细胞组上清液中IL-2和IFN-γ水平与CD20^-组相比明显升高。结论重组基因修饰的CD20靶向性T细胞构建成功。该T细胞在体外能特异性杀伤CD20^＋淋巴瘤细胞株。     

Cure of progressive murine leishmaniasis: interleukin 4 dominance is abolished by transient CD4(+) T cell depletion and T helper cell type 1-selective cytokine therapy.

Progressive infection with Leishmania major in susceptible BALB/c mice is mediated by interleukin (IL)-4-producing T helper cell type 2 (Th2) CD4(+) T cells that, once established, become resistant to Th1-deviating therapies with recombinant (r)IL-12 and/or neutralizing anti-IL-4 antibodies. We sought to restore protective immunity in advanced leishmaniasis by depletion of Th2-biased CD4(+) populations and by cytokine-directed reconstitution of Th1 cellular responses during lymphocyte recovery. Treatment with cytolytic GK1.5 anti-CD4 mAb alone did not reverse disease in 3 wk-infected BALB/c mice, but GK1.5 combined with anti-IL-4 antibody and intralesional rIL-12 cured cutaneous lesions in 80% of mice and established a Th1-polarized cytokine response to L. major antigen protective against reinfection. The curative effects of GK1.5 were not replaced by cytotoxic anti-CD8 monoclonal antibody 2.43 or nondepleting anti-CD4 mAb YTS177, confirming that depletion of CD4(+) cells was specific and essential for therapeutic effect. Finally, combined CD4(+) depletion and IL-4 neutralization were curative, indicating that neither increased parasite burden nor altered accessory cell function independently biased towards Th2 reconstitution in advanced leishmaniasis. Advanced leishmaniasis can be cured by T cell depletion and cytokine-directed recovery of Th1 cellular responses, suggesting novel interventions for other immune-mediated diseases and identifying distinct roles for CD4(+) T cell and non-T cell in the maintenance of Th2 and Th1 phenotypes.     

Alpha E beta 7 integrin interaction with E-cadherin promotes antitumor CTL activity by triggering lytic granule polarization and exocytosis

Various T cell adhesion molecules and their cognate receptors on target cells promote T cell receptor (TCR)-mediated cell killing. In this report, we demonstrate that the interaction of epithelial cell marker E-cadherin with integrin alpha(E)(CD103)beta(7), often expressed by tumor-infiltrating lymphocytes (TILs), plays a major role in effective tumor cell lysis. Indeed, we found that although tumor-specific CD103(+) TIL-derived cytotoxic T lymphocyte (CTL) clones are able to kill E-cadherin(+)/intercellular adhesion molecule 1(-) autologous tumor cells, CD103(-) peripheral blood lymphocyte (PBL)-derived counterparts are inefficient. This cell killing is abrogated after treatment of the TIL clones with a blocking anti-CD103 monoclonal antibody or after targeting E-cadherin in the tumor using ribonucleic acid interference. Confocal microscopy analysis also demonstrated that alpha(E)beta(7) is recruited at the immunological synapse and that its interaction with E-cadherin is required for cytolytic granule polarization and subsequent exocytosis. Moreover, we report that the CD103(-) profile, frequently observed in PBL-derived CTL clones and associated with poor cytotoxicity against the cognate tumor, is up-regulated upon TCR engagement and transforming growth factor beta1 treatment, resulting in strong potentiation of antitumor lytic function. Thus, CD8(+)/CD103(+) tumor-reactive T lymphocytes infiltrating epithelial tumors most likely play a major role in antitumor cytotoxic response through alpha(E)beta(7)-E-cadherin interactions.     

Injury to autologous normal tissues and tumors mediated by lymphokine-activated killer (LAK) cells generated in vitro from peripheral blood mononuclear cells of glioblastoma patients

Activation of peripheral blood mononuclear cells (PBMC) with IL-2 generates lymphokine-activated killer (LAK) cells that show a broad target cell range. In adoptive immunotherapy using in vitro-generated LAK cells, the intensity and specificity of their cytotoxic activity affect the prognosis of cancer patients. The present study was designed to examine the tumor-specific spectrum of T lymphocytes generated from the PBMC of patients with recurrent glioblastoma by in vitro propagation with IL-2 plus either soluble or solid-phase anti-CD3 monoclonal antibody (MAb) in short-term or long-term cultures. Both short-term and long-term culturing with solid-phase anti-CD3 MAb plus IL-2 yielded broad-reactivity CD8+ alphabetaT and gammadeltaT lymphocytes, both of which were non-MHC restricted, as shown by the fact that they were able to lyse autologous glioblastoma cells, MHC class I+II- allogeneic glioblastoma cells, and MHC class I-II-NK-sensitive K562 target cells. More importantly, these cells from patients failed to lyse fresh autologous PBMC. These results demonstrate that cells generated using this approach are non-MHC-restricted LAK cells and exhibit marked tumor specificity. In contrast, incubation with soluble anti-CD3 MAb generated T lymphocytes that after long-term culture, were either CD4+ or CD8+. These caused significant lysis of both allogeneic and autologous glioblastoma target cells, the extent of lysis being greater than that using cells produced by culturing with the solid-phase MAb. However, both the CD4+ and CD8+ cells also caused greater lysis of autologous normal PBMC, indicating that cells generated using this approach may cause significant adverse reactions in cancer patients if used for immunotherapy.     

CD4+CD25+ regulatory T cells suppress allograft rejection mediated by memory CD8+ T cells via a CD30-dependent mechanism

CD4(+)CD25(+) regulatory T (Treg) cells suppress naive T cell responses, prevent autoimmunity, and delay allograft rejection. It is not known, however, whether Treg cells suppress allograft rejection mediated by memory T cells, as the latter mount faster and stronger immune responses than their naive counterparts. Here we show that antigen-induced, but not naive, Treg cells suppress allograft rejection mediated by memory CD8(+) T cells. Suppression was allospecific, as Treg cells induced by third-party antigens did not delay allograft rejection. In vivo and in vitro analyses revealed that the apoptosis of allospecific memory CD8(+) T cells is significantly increased in the presence of antigen-induced Treg cells, while their proliferation remains unaffected. Importantly, neither suppression of allograft rejection nor enhanced apoptosis of memory CD8(+) T cells was observed when Treg cells lacked CD30 or when CD30 ligand-CD30 interaction was blocked with anti-CD30 ligand Ab. This study therefore provides direct evidence that pathogenic memory T cells are amenable to suppression in an antigen-specific manner and identifies CD30 as a molecule that is critical for the regulation of memory T cell responses.     

Concomitant tumor immunity to a poorly immunogenic melanoma is prevented by regulatory T cells

Concomitant tumor immunity describes immune responses in a host with a progressive tumor that rejects the same tumor at a remote site. In this work, concomitant tumor immunity was investigated in mice bearing poorly immunogenic B16 melanoma. Progression of B16 tumors did not spontaneously elicit concomitant immunity. However, depletion of CD4(+) T cells in tumor-bearing mice resulted in CD8(+) T cell-mediated rejection of challenge tumors given on day 6. Concomitant immunity was also elicited by treatment with cyclophosphamide or DTA-1 monoclonal antibody against the glucocorticoid-induced tumor necrosis factor receptor. Immunity elicited by B16 melanoma cross-reacted with a distinct syngeneic melanoma, but not with nonmelanoma tumors. Furthermore, CD8(+) T cells from mice with concomitant immunity specifically responded to major histocompatibility complex class I-restricted epitopes of two melanocyte differentiation antigens. RAG1(-/-) mice adoptively transferred with CD8(+) and CD4(+) T cells lacking the CD4(+)CD25(+) compartment mounted robust concomitant immunity, which was suppressed by readdition of CD4(+)CD25(+) cells. Naturally occurring CD4(+)CD25(+) T cells efficiently suppressed concomitant immunity mediated by previously activated CD8(+) T cells, demonstrating that precursor regulatory T cells in naive hosts give rise to effective suppressors. These results show that regulatory T cells are the major regulators of concomitant tumor immunity against this weakly immunogenic tumor.     

Induction of leukemic-cell-specific cytotoxic T lymphocytes by autologous monocyte-derived dendritic cells presenting leukemic cell antigens

Leukemic-dendritic cells (leukemic-DCs) have certain limitations, which include difficult generation in 30-40% of patients, and low levels of expression of several key molecules. Therefore, an alternative approach using monocyte-derived DCs pulsed with tumor antigens is required. We investigated the possibility of immunotherapy for AML using leukemic-cell-specific cytotoxic T lymphocytes that were stimulated in vitro by autologous DCs pulsed with tumor antigens. To generate DCs, CD14(+) cells were isolated from peripheral blood mononuclear cells using magnetic-activated cell sorting, and cultured in the presence of GM-CSF and IL-4. On day 6, maturation of DCs was induced by addition of cytokine cocktail (TNF-alpha, IL-1beta, IL-6, and prostaglandin E(2)) for 2 days, and then the mature DCs were pulsed with whole leukemic cell lysates or apoptotic leukemic cells. There were no differences in the phenotypic expressions of mature DCs generated by pulsing with or without leukemic antigens. The mature DCs pulsed with tumor cell lysates or apoptotic leukemic cells showed a higher allostimulatory capacity for allogeneic CD3(+) T cells as compared with mature non-pulsed DCs. Autologous CD3(+) T cells stimulated by the mature pulsed DCs showed more potent cytotoxic activities against autologous leukemic cells than those stimulated by mature non-pulsed DCs. These results suggest that use of DCs pulsed with leukemic cell lysates or apoptotic leukemic cells is a feasible alternative immunotherapeutic approach to overcome the limitations of leukemic-DCs for the treatment of AML patients.     

HIV-1 infection impairs cell cycle progression of CD4+ T cells without affecting early activation responses

Failure of CD4(+) T cells to proliferate in response to antigenic stimulation is a characteristic of HIV infection. Analysis of the proliferation defect has been hampered by an inability to identify CD4(+) cells with T cell receptor specificity for antigen. To focus only on cells that had been stimulated through the T cell receptor, CD4(+) T cells were stimulated with an anti-Vbeta3 Ab that activates approximately 3-5% of peripheral blood T cells. This approach revealed proliferation defects in cells from HIV-infected patients that were not appreciated using anti-CD3 Ab stimulation and provided the capacity to examine responses on a single cell basis. After anti-Vbeta3 Ab stimulation, CD4(+)Vbeta3(+) cells from HIV-infected patients demonstrated defects in expression of cell cycle-associated proteins, D-type cyclins, and cyclin A. However, the expression of early activation markers, CD69 and CD25, was not significantly impaired in cells from most patients. Thus, CD4(+) T cell proliferation failure in HIV disease is characterized by dysregulated activation that precludes cell cycle progression. This proliferation defect was most apparent in patients with diminished CD4(+) T cell numbers and higher plasma HIV RNA levels. CD4(+) T cell proliferation failure may be a key determinant of immune impairment in HIV disease.     

Urushiol (poison ivy)-triggered suppressor T cell clone generated from peripheral blood

Allergic contact dermatitis to Toxicodendron radicans (poison ivy) is mediated by the hapten urushiol. An urushiol-specific, interleukin 2 (IL-2)-dependent T cell clone (RLB9-7) was generated from the peripheral blood of a patient with a history of allergic contact dermatitis to T. radicans. This clone proliferated specifically to both leaf extract and pure urushiol. Although the clone had the phenotype CD3+CD4+CD8+, proliferation to antigen was blocked by anti-CD8 and anti-HLA-A, B, C, but not by anti-CD4, suggesting that CD4 was not functionally associated with the T cell receptor. Furthermore, studies with antigen-presenting cells from MHC-typed donors indicated that the clone was MHC class 1 restricted. RLB9-7 was WT31 positive, indicating it bears the alpha beta T cell receptor. The clone lacked significant natural killer cell activity and produced only low levels of IL-2 or gamma-interferon upon antigen stimulation. Addition of RLB9-7 to autologous peripheral blood mononuclear cells in the presence of urushiol inhibited the pokeweed mitogen-driven IgG synthesis. This suppression was resistant to irradiation (2,000 rad) and was not seen when RLB9-7 was added to allogeneic cells, even in the presence of irradiated autologous antigen-presenting cells, suggesting that suppression was MHC restricted and not mediated by nonspecific soluble factors. However, RLB9-7 cells in the presence of urushiol inhibited the synthesis of tetanus toxoid-specific IgG by autologous lymphocytes, indicating that the suppression, although triggered specifically by urushiol, was nonspecific.     

A cloned human T cell line cytotoxic for autologous and allogeneic B lymphoma cells

A human cytotoxic T cell clone (MWS-14) with auto-tumor reactivity was established in serum-free medium in a mixed tumor cell culture by repetitive stimulation with fresh autologous lymphoma cells. This clone and its subclones are of the T3+ T4+ T8- phenotype. They were strongly cytotoxic for the autologous lymphoma cells, whereas autologous PHA blasts were not killed. Analysis of the specificity of MWS-14, MWS-14-30, and MWS-14-34 indicated that these CTL clones were cytotoxic for 7/7 allogeneic lymphoma cells, whereas only 3/23 of normal and non-lymphoma cells were lysed. Blocking studies with monoclonal antibodies directed at MHC class I and class II antigens showed that this preferential, anti-lymphoma reactivity was not directed at HLA determinants. The anti-lymphoma activity is not due to an aspecific susceptibility of the lymphoma cells to lysis. In contrast to CTL clones specific for HLA antigens present on the lymphoma cells, T3 and T4 were not involved in the cytotoxic reaction of MWS-14 against the autologous lymphoma cells. The reactivity of this clone could be blocked by a monoclonal antibody directed at leukocyte function-associated antigen. It can be concluded from these results that these T4+ CTL clones recognize a determinant, which is preferentially expressed on autologous and allogeneic lymphoma cells.     

Specifically activated memory T cell subsets from cancer patients recognize and reject xenotransplanted autologous tumors

Bone marrow of breast cancer patients was found to contain CD8(+) T cells specific for peptides derived from breast cancer-associated proteins MUC1 and Her-2/neu. Most of these cells had a central or effector memory phenotype (CD45RA(-)CD62L(+) or CD45RA(-)CD62L(-), respectively). To test their in vivo function, we separated bone marrow-derived CD45RA(+) naive or CD45RA(-)CD45RO(+) memory T cells, stimulated them with autologous dendritic cells pulsed with tumor lysate, and transferred them into NOD/SCID mice bearing autologous breast tumors and normal skin transplants. CD45RA(-) memory but not CD45RA(+) naive T cells infiltrated autologous tumor but not skin tissues after the transfer. These tumor-infiltrating cells had a central or effector memory phenotype and produced perforin. Many of them expressed the P-selectin glycoprotein ligand 1 and were found around P-selectin(+) tumor endothelium. Tumor infiltration included cluster formation in tumor tissue by memory T cells with cotransferred dendritic cells. It was associated with the induction of tumor cell apoptosis and significant tumor reduction. We thus demonstrate selective homing of memory T cells to human tumors and suggest that tumor rejection is based on the recognition of tumor-associated antigens on tumor cells and dendritic cells by autologous specifically activated central and effector memory T cells.     

Antigen recognition by human T cell receptor gamma-positive lymphocytes. Specific lysis of allogeneic cells after activation in mixed lymphocyte culture

These experiments were designed to define the ability of human TCR-gamma+ cells to recognize allogeneic cells. TCR-gamma+-enriched populations were obtained by treating peripheral blood E-rosetting cells with anti-CD4 and anti-CD8 mAbs. The resulting populations were CD2+4-8- expressed variable proportions of CD3+ cells (40-90%), and did not react with the WT31 mAb, which is specific for a framework determinant of the alpha/beta heterodimer that serves as receptor for antigen on most human T lymphocytes. After mixed lymphocyte culture with irradiated allogeneic cells for 7 d and 3 additional days in rIL-2 (100 U/ml), cells underwent proliferation in three of five individuals tested. In addition, MLC-derived cells lysed 51Cr-labeled PHA-induced blasts derived from the allogeneic cells used as stimulator, but not allogeneic unrelated or autologous blast cells. No cytotoxicity against autologous or allogeneic target cells could be induced by culturing CD3+4-8-WT31- lymphocytes in MLC with irradiated autologous cells. Surface iodination of allogeneic MLC-activated CD3+4-8-WT31- cells followed by lysis in 1% digitonin and immunoprecipitation with anti-CD3 mAb indicated that the CD3-associated molecules consisted of a major 45-kD band and a minor band of 43 kD. Northern blot analysis showed that mRNA for the gamma chain was expressed at high levels, whereas mRNAs for alpha and beta chains were missing. These data support the notion that TCR-gamma rather than TCR-alpha/beta is expressed in allospecific CD3-4-8-WT31- cell populations. Clones were further derived from MLC-stimulated CD3+4-8-WT31- populations. All the seven clones studied in detail maintained the surface phenotype as well as the cytolytic pattern of the original MLC populations, thus only specific allogeneic PHA-induced blasts were lysed. NK-sensitive as well as NK-resistant tumor targets were variably susceptible to lysis; therefore, specific cytolytic activity against allogeneic cells was not necessarily linked to the expression of MHC-nonrestricted cytotoxicity against tumor cells.     

CTLA4 blockade maximizes antitumor T-cell activation by dendritic cells presenting idiotype protein or opsonized anti-CD20 antibody-coated lymphoma cells

CTLA4 is a negative regulator of the costimulatory signals induced by the interaction of CD28 on T cells and B7 on dendritic cells (DCs). Antibodies (Abs) against CTLA4 can block its function and increase the activation of T cells primed to recognize antigens. The effect of CTLA4 blockade on the cross-presentation of tumor antigens by DCs to T cells was examined. Immune T cells and DC precursors were collected from patients receiving idiotype protein-pulsed DC vaccines, exposed to antigen, and examined for antitumor activity by measuring intracellular cytokine production by FACS. Idiotype-specific activation occurred in CD8+ and CD4+ T-cell populations and was up to 58 fold higher with CTLA4 blockade. These T cells could be expanded quickly and maintained tumor cytolytic activity. T-cell responses to whole tumor cell-pulsed DCs were then examined. DCs contain Fc receptors and efficiently phagocytose lymphoma cells when coated with opsonizing anti-CD20 Abs. Within a few hours, DCs ingested tumor cells and labeled proteins were observed in the cytoplasm. When anti-CD20 Ab-coated tumor-pulsed DCs were used in combination with CTLA4 blockade, up to 15 fold higher activation of Id-specific CD8+ and 3 fold higher CD4+ T cells resulted. Thus, CTLA4 blockade can enhance the measurement of Ag-specific T-cell responses and the expansion of T cells for clinical studies. In addition, the combination of CTLA4 blockade and Ab targeting of tumor to DCs is an effective method for the cross-presentation of tumor cell antigens.     

CD30-mediated signaling promotes the development of human T helper type 2-like T cells

We have recently shown that CD30, a member of the tumor necrosis factor/nerve growth factor receptor superfamily, is preferentially expressed by human T cell clones producing T helper (Th) type 2 cytokines. We report here that costimulation with an agonistic anti- CD30 monoclonal antibody enhanced antigen (Ag)-induced proliferation and cytokine secretion by established human Th2 and Th0 clones. Moreover, costimulation of peripheral blood mononuclear cells with the same anti-CD30 monoclonal antibody resulted in the preferential development of Ag-specific T cell lines and clones showing a Th2-like profile of cytokine secretion. In contrast, early blockade in bulk culture of CD30 ligand-CD30 interaction shifted the development of Ag- specific T cells towards the opposite (Th1-like) phenotype. Taken together, these data suggest that CD30 triggering of activated Th cells by CD30 ligand-expressing Ag-presenting cells may represent an important costimulatory signaling for the development of Th2-type responses.