Adoptive T cell therapy of solid cancers

The development of immune-based approaches for the treatment of cancer has been actively investigated for many years. One strategy that has emerged as a potentially effective strategy for the treatment of aggressive established malignancies is adoptive T cell therapy. The power of this approach has been repeatedly observed in preclinical animal models. However, moving from homogeneous animal models to the heterogeneous human clinical setting has been very difficult. It is only in recent times that we have been able to pinpoint the problems of the clinical translation of adoptive T cell therapy. Some of the major problems are sources of tumor-specific T cells, ex vivo expansion, persistence, and anti-tumor activity. This review overviews the nature of these problems and some of the emerging solutions. This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2004 (PIVAC 4)”, held in Freudenstadt-Lauterbad, Black Forest, Germany, on 22–25 September 2004 Grant support: This grant was supported by K01-CA100764 (KLK) and R01-CA85374 (MLD)     

A rational relationship: Oncolytic virus vaccines as functional partners for adoptive T cell therapy

Tumours employ a variety of immune-evasion and suppression mechanisms to impair development of functional tumor-specific T cells and subvert T cell-mediated immunity in the tumour microenvironment. Adoptive T cell therapy (ACT) aims to overcome these barriers and overwhelm tumor defenses with a bolus of T cells that were selectively expanded ex vivo. Although this strategy has been effective in liquid tumors and melanomas, many tumors appear to be resistant to ACT. Several factors are thought to play into this resistance, including poor engraftment and persistence of transferred cells, tumour cell heterogeneity and antigen loss, poor immune cell recruitment and infiltration into the tumour, and susceptibility to local immunosuppression in the tumor microenvironment. Oncolytic viruses (OV) have been identified as powerful stimulators of the anti-tumour immune response. As such, OVs are inherently well-positioned to act in synergy with ACT to bolster the anti-tumour T cell response. Further, OV vaccines, wherein tumour-associated antigens are encoded into the viral backbone, have proven to be remarkable in boosting antigen-specific T cell response. Pre-clinical studies have revealed remarkable therapeutic outcomes when OV vaccines are paired with ACT. In this scenario, OV vaccines are thought to function in a “push and pull” manner, where push refers to expanding T cells in the periphery and pull refers to recruiting those cells into the tumour that has been rendered amenable to T cell attack by the actions of the OV. In this review, we discuss barriers that limit eradication of tumors by T cells, highlight attributes of OVs that break down these barriers and present strategies for rational combinations of ACT with OV vaccines.     

Adoptive-transfer therapy of tumors with the tumor-specific primary cytotoxic T cells induced in vitro with the B7.1-transduced MCA205 cell line

We show that the tumor-specific primary cytotoxic T lymphocytes (CTL) induced in vitro with the MCA205 fibrosarcoma cells transduced with the B7.1 (CD80) gene are highly effective in adoptive-transfer therapy of the parental tumors. The MCA205 fibrosarcoma cell line was transduced with the retroviral vectors encoding the B7.1 gene and tested for their efficiency as stimulators in short-term (5 days) mixed lymphocyte/tumor cell cultures with highly purified syngenic, unprimed T cells as responders. The induction of the CTL required the presence of a low dose of interleukin-2 (25 U/ml). The injection of the CTL prevented colony formation by the intravenously injected tumor cells in a lung colonization assay in which the CTL were injected after inoculation of tumor cells. We also showed that the adoptive transfer of the same T cells was effective in delaying the growth of the subcutaneously injected tumor cells. These results imply that the short-term mixed lymphocyte/tumor cell culture with the tumor cells transduced with the gene for the B7.1 costimulatory molecule is potentially a good source of CTL for adoptive-transfer therapy of tumors. Received: 30 June 1998 / Accepted: 5 August 1998     

Gene gun application in the generation of effector T cells for adoptive immunotherapy

We utilized the gene gun to transfect subcutaneous D5 melanoma and MT-901 mammary carcinoma tumors in situ with a granulocyte/macrophage-colony-stimulating factor (GM-CSF) plasmid complexed to gold particles. There was diminished tumor growth following bombardment with GM-CSF plasmid, which was apparent only during the period of administration. Transgenic GM-CSF was produced by the skin overlying the tumors and not by the tumors themselves. GM-CSF plasmid bombardment resulted in increased cell yields within tumor-draining lymph nodes (TDLN) with at least a 12-fold increase in the percentage of dendritic cells (8.9%) compared to controls (0.7%). Secondarily activated TDLN cells from animals transfected with GM-CSF demonstrated enhanced cytokine release (interferon γ, GM-CSF and interleukin-10) in response to tumor stimulator cells compared to controls, and had an increased capacity to mediate tumor regression in adoptive immunotherapy. There was a small, but detectable, non-specific immune adjuvant effect observed with gold particle bombardment alone, which was less than with GM-CSF plasmid. The adjuvant effect of GM-CSF plasmid required peri-tumoral transgene expression since gene bombardment away from the tumor was ineffective. Received: 27 April 1999 / Accepted: 27 August 1999     

Safety profile and anti-tumor effects of adoptive immunotherapy using gamma-delta T cells against advanced renal cell carcinoma: a pilot study

Purpose Although various types of immunotherapy have been used to improve the prognosis of patients with advanced renal cell carcinoma (RCC), adoptive immunotherapy using gamma-delta (γδ) T cells has not yet been tried. In this study, we designed a pilot study of adoptive immunotherapy using in vitro activated γδ T cells against advanced RCC to evaluate the safety profile and possible anti-tumor effects of this study. Experimental design Patients with advanced RCC after radical nephrectomy were administered via intravenous infusion in vitro-activated autologous γδ T cells every week or every 2 weeks, 6–12 times, with 70 JRU of teceleukin. Adverse events, anti-tumor effects and immunomonitoring were assessed. The anti-tumor effects were evaluated according to tumor doubling time (DT) by computed tomography (CT) and immunomonitoring was performed by flow cytometric analysis. Results Seven advanced RCC patients were entered in this study. The most common adverse events were fever, general fatigue and elevation of hepatobiliary enzymes, but no severe adverse events were seen. Prolongation of tumor DT was seen in three out of five patients; these three patients showed an increase in the number of γδ T cells in peripheral blood and also a high response to the antigen in vitro. Conclusions The results indicated that adoptive immunotherapy using in vitro-activated autologous γδ T cells was well tolerated and induced anti-tumor effects.     

Adjuvant effect of anti-4-1BB mAb administration in adoptive T cell therapy of cancer

Administration of anti-4-1BB mAb has been found to be a potent adjuvant when combined with other therapeutic approaches, e.g. chemotherapy, cytokine therapies, anti-OX40 therapy, and peptide or DC vaccines. However, the adjuvant effect of anti-4-1BB mAb administration in adoptive T cell therapy of cancer has not been fully evaluated. In this report, effector T cells were generated in vitro by anti-CD3/anti-CD28 activation of tumor-draining lymph node (TDLN) cells and used in an adoptive immunotherapy model. While T cells or anti-4-1BB alone showed no therapeutic efficacy in mice bearing macroscopic 10-day pulmonary metastases, T cells plus anti-4-1BB mediated significant tumor regression in an anti-4-1BB dose dependent manner. Mice bearing microscopic 3-day lung metastases treated with T cells alone demonstrated tumor regression which was significantly enhanced by anti-4-1BB administration. NK cell depletion abrogated the augmented therapeutic efficacy rendered by anti-4-1BB. Cell transfer between congenic hosts demonstrated that anti-4-1BB administration increased the survival of adoptively transferred TDLN cells. Using STAT4(-/-) mice, we found that modulated IFN gamma secretion in wt TDLN cells after anti-CD3/CD28/4-1BB activation in vitro was lost in similarly stimulated STAT4(-/-) TDLN cells. Additionally, anti-4-1BB administration failed to augment the therapeutic efficacy of T cell therapy in STAT4(-/-) mice. Together, these results indicate that administered anti-4-1BB mAb can serve as an effective adjuvant to augment the antitumor reactivity of adoptively transferred T cells by recruiting the host NK cells; increasing the persistence of infused effector T cells, and modulating the STAT4 molecular signaling pathway.     

Enhancement of T cell localization in mammary tumors through transient inhibition of T cell myosin function

Adoptive immunotherapy is hampered by poor lymphocyte localization in tumors. The polarized, adhesive phenotype of activated lymphocytes may contribute to this problem by making the cells prone to trapping and damage in pulmonary microvasculature. We found that transient inhibition of T cell polarization prior to i.v. infusion reduces trapping and improves tumor localization. Activated T cells were rendered nonpolar and nonadhesive by treatment with myosin light-chain kinase inhibitor ML-7. Polarity, adhesiveness, and motility recovered by 6 h after treatment, cytotoxicity, and proliferation by 24 h. ErbB2-specific T cells were infused i.v. into mice bearing ErbB2-expressing mammary tumors. ML-7 pre-treatment reduced T cell arrest in lungs by a factor of eight, improved tumor localization by 4-fold, and increased lymph node homing. Although this improvement alone proved insufficient to alter outcome in an immunotherapy experiment, this study indicates that cytoskeletal modification is a promising strategy for altering the trafficking of infused lymphocytes.     

TCR transgenes and transgene cassettes for TCR gene therapy: status in 2008

The genetic introduction of T cell receptor genes into T cells has been developed over the past decade as a strategy to induce defined antigen-specific T cell immunity. With the potential value of TCR gene therapy well-established in murine models and the feasibility of infusion of TCR-modified autologous T cells shown in a first phase I trial, the next key step will be to transform TCR gene transfer from an experimental technique into a robust clinical strategy. In this review, we discuss the different properties of the TCR transgene and transgene cassette that can strongly affect both the efficacy and the safety of TCR gene transfer. This paper is a focussed research review based on a presentation given at the sixth annual meeting of the Association for Immunotherapy of Cancer (CIMT), held in Mainz, Germany,15–16 May 2008.     

Novel antigens of CAR T cell therapy: New roads; old destination

Chimeric antigen receptor T cell (CAR-T) therapy has so far proved itself as a reliable therapeutic option for the treatment of relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), multiple myeloma (MM), and mantle cell lymphoma (MCL). However, this picture is not as colorful when it comes to the treatment of solid tumors mainly due to the lack of definitive tumor antigens, as well as the immunosuppressive tumor microenvironments and poor CAR-T infiltration. The recent developments in bioinformatics and cell biology, such as single-cell RNA sequencing, have offered silver linings in the subject of tumor antigen discovery. In the current review, we summarize the development of some CAR-T therapies that target novel tumor antigens, rather than the traditionally CAR-T-targeted ones, and briefly discuss the clinical antitumor achievements of those evaluated in patients, so far. Furthermore, we propose some tumor antigens that might someday be therapeutically beneficial while targeted by CAR-Ts based on the experimental evaluations of their specific monoclonal antibodies.     

Requirement of monocytes and T-helper cells during development of tumor cell cytotoxicity in targeted T cells

In cocultures of human plancental alkaline phosphatase(PLAP)-positive MO₄ tumor cells and human peripheral blood mononuclear cells (PBMC), also containing a heteroconjugate (7E8-OKT3) synthesized between the anti-PLAP monoclonal antibody 7E8 and the anti-CD3 antibody OKT3, and supplemented with low levels of recombinant interleukin-2 (rIL-2), T cells are progressively activated, resulting in tumor cell lysis. To unravel the contribution of PBMC subsets to the generation of this targetable cytotoxicity, PBMC subsets were studied after their isolation by cell sorting, either from fresh PBMC or from PBMC peractivated with OKTe3 and rIL-2. Whereas no targetable cytotoxicity was found in Fc-receptor-bearing CD3-cells, tumor cells were lysed by CD3⁺ T cells (mostly CD8⁺) isolated from pre-activated PBMC. When isolated from fresh PBMC, neither the CD8⁺ T cell subset, nor the total CD3⁺ T cell population developed significant targetable cytotoxicity, even in the presence of rIL-2. Thus, additional cell types are essential for the CD8⁺ T cell activation. Indeed. CD4⁺ T cells isolated from pre-activated but not from fresh PBMC were capable of eliciting cytotoxicity in fresh CD8⁺ T cells. The non-targeted monocytes were found to be the activators of the CD4⁺ T cells. In summary, targeting T cells to the surface of a tumor cell is not sufficientper se to achieve activation and lysis. The progressive tumor cell lysis by targeted T cells seems to be initiated by non-targeted monocytes activating CD4⁺ T cells, these cells in turn promoting CD8⁺ T cell activation, necessary for the development of cytotoxicity.     

Targeting stromal cell sialylation reverses T cell-mediated immunosuppression in the tumor microenvironment

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Adoptive transfer of T<Subscript>reg</Subscript> depleted autologous T cells in advanced renal cell carcinoma

Purpose

CD4⁺CD25⁺ regulatory T (T_reg) cells are present in increased numbers in patients with advanced cancer and CD25⁺ T cell depletion potentiates tumour immunity in animal models. The aim of this study was to assess the feasibility and safety of adoptive transfer of CD25⁺ depleted autologous T cells in patients with advanced renal cell carcinoma and to examine resulting changes in lymphocyte subsets.

Patients and methods

Six patients with advanced renal cell carcinoma underwent leukapheresis followed by conditioning chemotherapy with cyclophosphamide and fludarabine. The autologous leukapheresis product was depleted of CD25⁺ cells using CliniMACS^® System then re-infused into the patient.

Results

Efficient CD25⁺ depletion from all leukapheresis products was achieved and 0.55–5.87 × 10⁷/kg CD3⁺ cells were re-infused. Chemotherapy related haematological toxicity was observed, but blood counts recovered in all patients allowing discharge after a mean inpatient stay of 21 days. One patient subsequently developed a rapidly progressive neurological syndrome. A transient reduction in CD25⁺ subset was noted in the peripheral blood of 5 out of 6 patients with evidence of increased T cell responses to PHA in 4 out of 6 patients. One patient showed increased specific proliferative responses to the tumour associated antigen h5T4 coinciding with the nadir of T_reg cells.

Conclusions

Given the transient nature of the reduction in CD25⁺ subset and the observed toxicity there is a need to explore further strategies to improve the safety and efficacy of this approach. Nevertheless, the results provide proof of concept in potentiation of tumour antigen T cell responses when T_reg cell levels are depleted.

     

Anti-tumor activity and trafficking of self, tumor-specific T cells against tumors located in the brain

It is commonly believed that T cells have difficulty reaching tumors located in the brain due to the presumed "immune privilege" of the central nervous system (CNS). Therefore, we studied the biodistribution and anti-tumor activity of adoptively transferred T cells specific for an endogenous tumor-associated antigen (TAA), gp100, expressed by tumors implanted in the brain. Mice with pre-established intracranial (i.c.) tumors underwent total body irradiation (TBI) to induce transient lymphopenia, followed by the adoptive transfer of gp100(25-33)-specific CD8+ T cells (Pmel-1). Pmel-1 cells were transduced to express the bioluminescent imaging (BLI) gene luciferase. Following adoptive transfer, recipient mice were vaccinated with hgp100(25-33) peptide-pulsed dendritic cells (hgp100(25-33)/DC) and systemic interleukin 2 (IL-2). This treatment regimen resulted in significant reduction in tumor size and extended survival. Imaging of T cell trafficking demonstrated early accumulation of transduced T cells in lymph nodes draining the hgp100(25-33)/DC vaccination sites, the spleen and the cervical lymph nodes draining the CNS tumor. Subsequently, transduced T cells accumulated in the bone marrow and brain tumor. BLI could also detect significant differences in the expansion of gp100-specific CD8+ T cells in the treatment group compared with mice that did not receive either DC vaccination or IL-2. These differences in BLI correlated with the differences seen both in survival and tumor infiltrating lymphocytes (TIL). These studies demonstrate that peripheral tolerance to endogenous TAA can be overcome to treat tumors in the brain and suggest a novel trafficking paradigm for the homing of tumor-specific T cells that target CNS tumors.     

Combined alpha tumor necrosis factor gene therapy and engineered dendritic cell vaccine in combating well-established tumors

BACKGROUND: Although current immunotherapeutic strategies including adenovirus (AdV)-mediated gene therapy and dendritic cell (DC) vaccine can all stimulate antitumor cytotoxic T lymphocyte (CLT) responses, their therapeutic efficiency has still been limited to generation of prophylactic antitumor immunity against re-challenge with the parental tumor cells or growth inhibition of small tumors in vivo. However, it is the well-established tumors in animal models that mimic clinical patients with existing tumor burdens. Alpha tumor necrosis factor (TNF-alpha) is a multifunctional and immunoregulatory cytokine that induces antitumor activity and activates immune cells such as DCs and T cells. We hypothesized that a combined immunotherapy including gene therapy and DC vaccine would have some advantages over each modality administered as a monotherapy. METHODS: We investigated the antitumor immunotherapeutic efficiency of gene therapy by intratumoral injection of AdVTNF-alpha and DC vaccine using subcutaneous injection of TNF-alpha-gene-engineered DC(TNF-alpha) cells, and further developed a combined AdV-mediated TNF-alpha-gene therapy and TNF-alpha-gene-engineered DC(TNF-alpha) vaccine in combating well-established MO4 tumors expressing the ovalbumin (OVA) gene in an animal model. RESULTS: Our data show that vaccination of DC(TNF-alpha) cells pulsed with the OVA I peptide can (i) stimulate type 1 immune response with enhanced antitumor CTL activities, (ii) induce protective immunity against challenge of 5 x 10(5) MO4 tumor cells, and (iii) reduce growth of the small (3-4 mm in diameter), but not large, established MO4 tumors (6-8 mm in diameter). Our data also show that AdVTNF-alpha-mediated gene therapy can completely eradicate small tumors in 6 out of 8 (75%) mice due to the extensive tumor necrosis formation, but not the large tumors (0%). Interestingly, a combined AdVTNF-alpha-mediated gene therapy and TNF-alpha-gene-engineered DC(TNF-alpha) vaccine is able to cure 3 out of 8 (38%) mice bearing large MO4 tumors, indicating that the combined immunotherapy strategy is much more efficient in combating well-established tumors than monotherapy of either gene therapy or DC vaccine alone. CONCLUSIONS: This novel combined immunotherapy may become a tool of considerable conceptual interest in the implementation of future clinical objectives.     

Rapid accumulation of adoptively transferred CD8+ T cells at the tumor site is associated with long-term control of SV40 T antigen-induced tumors

We previously established a model to study CD8⁺ T cell (T_CD8)-based adoptive immunotherapy of cancer using line SV11 mice that develop choroid plexus tumors in the brain due to transgenic expression of Simian Virus 40 large T antigen (Tag). These mice are tolerant to the three dominant T_CD8-recognized Tag epitopes I, II/III and IV. However, adoptive transfer of spleen cells from naïve C57BL/6 (B6) mice prolongs SV11 survival following T_CD8 priming against the endogenous Tag epitope IV. In addition, survival of SV11 mice is dramatically increased following transfer of lymphocytes from Tag-immune B6 mice. In the current study, we compared the kinetics and magnitude of Tag-specific T_CD8 accumulation at the tumor site following adoptive transfer with a high dose of either Tag-immune or naïve donor cells or decreasing doses of Tag-immune lymphocytes. Following adoptive transfer of Tag-immune cells, epitope I- and IV-specific T_CD8 accumulated to high levels in the brain of SV11 mice, peaking at 5–7 days, while epitope IV-specific T_CD8 derived from naïve donors required three weeks to achieve peak levels. A similar delay in the peak of epitope IV-specific T_CD8 accumulation was observed when tenfold fewer Tag-immune donor cells were administered, reducing control of tumor progression. These results suggest that efficient and prolonged control of established autochthonous tumors is associated with high-level early accumulation of adoptively transferred T cells. We also provide evidence that although multiple specificities are represented in the Tag immune donor lymphocytes, epitope IV-specific donor T_CD8 play a predominant role in control of tumor growth.     

Induction of cytotoxic T cells and their antitumor activity in mice transgenic for carcinoembryonic antigen

In order to develop immunotherapy strategies that are based on eliciting immune responsiveness to the self-antigen, human carcinoembryonic antigen (CEA), we examined whether cytotoxic T lymphocyte (CTL) activity against CEA could be elicited in CEA-transgenic and nontransgenic mice. CEA-transgenic [C57BL/6-TGN(CEAGe)18FJP] and nontransgenic mice were primed with CEA-transfected syngeneic fibroblasts in combination with Corynebacterium parvum. Spleen cells from immunized mice were cultured with irradiated syngeneic MC-38 colon carcinoma cells transfected with CEA (MC-38.CEA) as stimulators prior to the measurement of CTL activity. Primed nontransgenic spleen cells showed augmented CTL activity against MC-38.CEA cells as compared with control parental MC-38 cells, nontransfected or transfected with vector only. Moreover, primed CEA transgenic spleen cells showed augmented CTL activity against MC-38.CEA cells that was similar to that observed in nontransgenic mice. All CTL clones derived from either transgenic or nontransgenic mice showed cross-reactivity with MC-38 cells expressing the CEA-related antigen, nonspecific cross-reacting antigen, but not biliary glycoprotein. CEA-specific CTL clones were not identified. Adoptive transfer of cloned CTL resulted in inhibition of MC-38.CEA but not MC-38.BGP tumor growth. Tumor cures were elicited in mice treated with a combination of cloned CTL and cyclophosphamide. Histopathological examination of CEA-expressing colons from either immunized mice or recipients of cloned CTL did not reveal any autoimmune reactions. These studies demonstrate that CTL recognizing cross-reactive class I epitopes on the CEA molecule can be induced in transgenic mice. The expression of these epitopes on tumor cells creates effective targets for CTL in vivo without inducing adverse reactions in CEA-expressing normal tissues. Since anti-CEA CTL have been generated in humans, CEA-transgenic mice may be a useful model to study vaccines that are based on CTL effector mechanisms. Received: 7 January 2000 / Accepted: 8 March 2000     

Gene-modified T cells for adoptive immunotherapy of renal cell cancer maintain transgene-specific immune functions in vivo

Background We have treated three patients with carboxy-anhydrase-IX (CAIX) positive metastatic renal cell cancer (RCC) by adoptive transfer of autologous T-cells that had been gene-transduced to express a single-chain antibody-G250 chimeric receptor [scFv(G250)], and encountered liver toxicity necessitating adaptation of the treatment protocol. Here, we investigate whether or not the in vivo activity of the infused scFv(G250)⁺ T cells is reflected by changes of selected immune parameters measured in peripheral blood. Methods ScFv(G250)-chimeric receptor-mediated functions of peripheral blood mononuclear cells (PBMC) obtained from three patients during and after treatment were compared to the same functions of scFv(G250)⁺ T lymphocytes prior to infusion, and were correlated with plasma cytokine levels. Results Prior to infusion, scFv(G250)⁺ T lymphocytes showed in vitro high levels of scFv(G250)-chimeric receptor-mediated functions such as killing of CAIX⁺ RCC cell lines and cytokine production upon exposure to these cells. High levels of IFN-γ were produced, whilst production of TNF-α, interleukin-4 (IL-4), IL-5 and IL-10 was variable and to lower levels, and that of IL-2 virtually absent. PBMC taken from patients during therapy showed lower levels of in vitro scFv(G250)-receptor-mediated functions as compared to pre-infusion, whilst IFN-γ was the only detectable cytokine upon in vitro PBMC exposure to CAIX. During treatment, plasma levels of IFN-γ increased only in the patient with the most prominent liver toxicity. IL-5 plasma levels increased transiently during treatment in all patients, which may have been triggered by the co-administration of IL-2. Conclusion ScFv(G250)-receptor-mediated functions of the scFv(G250)⁺ T lymphocytes are, by and large, preserved in vivo upon administration, and may be reflected by fluctuations in plasma IFN-γ levels.     

Therapeutic use of a long-term cytotoxic T cell line recognizing a common tumour-associated antigen: The pattern of in vitro reactivity predicts the in vivo effect on different tumours

Summary A long-term-cultured cytotoxic T lymphocyte (CTL) line (E/88) was obtained from splenic lymphocytes of BALB/c (H-2 ^d) mice bearing the weakly immunogenic colonic carcinoma C26. This line was shown to be /TCR⁺V6⁺CD3⁺CD8⁺CD4^– and to recognize a common tumour-associated antigen on syngeneic carcinomas and sarcomas in a major-histocompatibility—complex-restricted and T-cell-receptor(TCR)-mediated fashion. The assessment of cytotoxic activity on a panel of 30 normal and neoplastic target cells of differing etiology and histotype showed that E/88 CTL lysed syngeneic colon carcinomas and some fibrosarcomas but not leukemias, lymphomas or mammary carcinomas. Clones derived from the E/88 line exhibited the same lytic pattern. Moreover, anti-T3, anti-Lyt2.2, anti-/TCR and anti-V6 mAbs as well as anti-H-2^d antisera abolished cytotoxicity when used in blocking experiments. The therapeutic activity of E/88 CTL upon in vivo transfer was assessed in mice bearing either experimental or spontaneous metastases of C26. In both models therapy with E/88 lymphocytes in combination or not with interleukin-2 was highly effective. Adoptive immunotherapy carried out with two clones obtained from line E/88 showed comparable therapeutic effects. In addition, treatment of syngeneic mice bearing experimental metastases of in vitro E/88-lysable or E/88-resistant tumours, showed that E/88 CTL can eradicate metastases of the former but not of the latter neoplasms. These data indicate that long-term CTL lines recognizing common tumour-associated antigens can be derived from tumourbearing animals and used in adoptive immunotherapy of tumours previously shown to be lysed in vitro by these effectors.