Establishment of cytotoxic T-cell clones specific for cells infected with mouse hepatitis virus.

Mouse hepatitis virus (MHV)-specific T-lymphocyte clones were established from MHV-infected BALB/c mice. They expressed Thy1 and Lyt2 antigens but lacked L3T4 and NK1 antigens. The clones killed MHV-infected but not uninfected or influenza virus-infected J774.1 cells. The specificity was further defined by a cold-target competition test.     

In vitro induction of tumor-specific immunity. IV. Specific adoptive immunotherapy with cytotoxic T cells induced in vitro to plasmacytoma antigens

Summary Specifically activated cytotoxic T cells (CL's) were raised by cocultivation in vitro of normal spleen cells with syngeneic plasmacytoma cells. These CL's were fully active both in vitro in lysing ⁵¹Cr-labelled tumor cells and in in vivo Winn assays, in inhibiting the growth of tumor cells in syngeneic mice when inoculated admixed with the tumor cells, even at ratios of 2 CL's to 1 tumor cell. However the same CL populations were only marginally effective in an immunotherapy model in which CL's were injected intravenously and tumor cells subcutaneously or intramuscularly (at a ratio of up to 100 CL/1 tumor cell). It is suggested that the in vitro conditions alter cell surface properties, thereby interfering with normal cell traffic, and that if this problem can be overcome, in vitro educated CL's may constitute a potential source of activated cells for human tumor immunotherapy.National Health and Medical Research Council postgraduate research scholar     

Generation and ex vivo expansion of HTLV-1 specific CD8+ cytotoxic T-lymphocytes for adoptive immunotherapy

CD8(+) cytotoxic T-lymphocytes (CTLs) have been proven, in multiple animal models, to be the most powerful antiviral and antitumor components of the immune system. We have developed a protocol to activate and expand tumor and virus peptide-specific CD8(+) T-lymphocytes from the peripheral blood of healthy, human trophic leukemia virus-1 (HTLV-1) seronegative human leucocyte antigen (HLA)-A*0201 individuals. A combination of density-based separation and culture conditions was employed to isolate dendritic cells (DCs), which are the most potent antigen-presenting cells (APCs), and T-lymphocytes. The DCs were pulsed with HLA-A*0201 binding peptides and cultured with autologous T-lymphocytes to generate peptide-specific CTLs. The CTLs were generated against a nine-amino-acid peptide from the Tax protein of HTLV-1. The CTLs were expanded according to a restimulation schedule employing peptide-pulsed autologous monocytes and low-dose interleukin-2 (IL-2) to numbers in excess of 100 x 10(6) cells following 5 weeks of culture. Expanded cells contained primarily CD3(+) T-cells, of which CD8(+) T-lymphocytes constituted greater than two-thirds of the cell population. Obtained CTLs exhibited potent antigen-specific lysis of peptide-pulsed target cells in a dose-dependent fashion in in vitro (51)Cr release cytotoxicity assay. This antigen-specific killing was shown to be HLA class I restricted and mediated by CD8(+) T-lymphocytes. Since the T-lymphocytes were obtained from HTLV-1 seronegative donors, the generation of peptide-specific CTLs represents reliable and reproducible elicitation of a primary immune response in vitro against naive antigens and subsequent expansion of generated CTLs for adoptive immunotherapy. (c) 1996 John Wiley & Sons, Inc.     

Expansion of tumor-specific cytolytic T lymphocytes using in vitro restimulation with tumor-specific transplantation antigen

Tumor-specific T lymphocytes (CTL) induced by in vivo immunization of C3H/HeJ mice with the syngeneic methylcholanthrene (MCA)-induced fibrosarcoma MCA-F were expanded in vitro by restimulation with 1-butanol-extracted, isoelectrophoretically purified, tumor-specific transplantation antigen (TSTA) in combination with purified rat interleukin-2 (IL-2) and fresh, syngeneic, 2000-R-irradiated, adherent splenic antigen-presenting cells (APC). The cultured immune T-cell population, containing 40–55% Lyt 2⁺ and 40–60% L3T4⁺ cells, displayed TSTA-specific proliferative and cytotoxic activities in vitro. The expanded T cells appear to recognize butanol-extracted TSTA in association with specific H-2 class I antigens, as revealed by the benefit of syngeneic over allogeneic cells as APC and by the adverse effect of depletion using anti-H-2K, but not anti-Ia, monoclonal antibodies. In adoptive transfer assays in vitro, expanded T cells specifically neutralize homotypic, but not heterotypic, tumor growth in vivo. Based upon the effects of depletion of T-lymphocyte subpopulations using monoclonal antibodies, the Lyt 2⁺ cytotoxic T lymphocytes (CTL) appear to display greater in vivo neutralizing activity than L3T4⁺ T cells. Thus in vitro stimulation of in vivo-immunized T cells, using butanol-extracted TSTA in combination with IL-2 and syngeneic APC, expands tumor-specific CTL.     

GMP production of anti-tumor cytotoxic T-cell lines for adoptive T-cell therapy in patients with solid neoplasia

BACKGROUND: The adoptive transfer of ex vivo-induced tumor-specific T-cell lines provides a promising approach for cancer immunotherapy. We have demonstrated previously the feasibility of inducing in vitro long-term anti-tumor cytotoxic T-cell (CTL) lines directed against different types of solid tumors derived from both autologous and allogeneic PBMC. We have now investigated the possibility of producing large amounts of autologous anti-tumor CTL, in compliance with good manufacturing practices, for in vivo use. METHODS: Four patients with advanced solid tumors (two sarcoma, one renal cell cancer and one ovarian cancer), who had received several lines of anticancer therapy, were enrolled. For anti-tumor CTL induction, patient-derived CD8-enriched PBMC were stimulated with DC pulsed with apoptotic autologous tumor cells (TC) as the source of tumor Ag. CTL were then restimulated in the presence of TC and expanded in an Ag-independent way. RESULTS: Large amounts of anti-tumor CTL (range 14-20 x 10(9)), which displayed high levels of cytotoxic activity against autologous TC, were obtained in all patients by means of two-three rounds of tumor-specific stimulation and two rounds of Ag-independent expansion, even when a very low number of viable TC was available. More than 90% of effector cells were CD3(+) CD8(+) T cells, while CD4(+) T lymphocytes and/or NK cells were less than 10%. DISCUSSION: Our results demonstrate the feasibility of obtaining large quantities of anti-tumor specific CTL suitable for adoptive immunotherapy approaches.     

Feasibility and safety of adoptive immunotherapy with ex vivo-generated autologous, cytotoxic T lymphocytes in patients with solid tumor

Background aimsAdoptive T-cell therapy with tumor-specific T cells has emerged as a potentially useful approach for treating patients with advanced malignancies. We have demonstrated previously the feasibility of obtaining large numbers of autologous anti-tumor-specific cytotoxic T lymphocytes (CTL) generated by stimulation of patients' peripheral blood mononuclear cells with dendritic cells pulsed with apoptotic tumor cellsMethodsSix patients with progressing metastatic solid tumors (one renal cell carcinoma, two ovarian cancers, two extraosseous peripheral neuroectodermal tumors, one soft tissue sarcoma) not eligible for conventional therapies were treated with adoptive immunotherapy. Anti-tumor CTL, proven to be reactive in vitro against patient tumor cells, but not against normal cells, were infused following lymphodepleting chemotherapy administered to favor T-cell proliferation in vivo.ResultsPatients received a median of nine CTL infusions (range 2–19). The median number of CTL administered per infusion was 11 × 10⁸ (range 1–55 × 10⁸). No patient experienced acute or late adverse events related to CTL infusion, even when large numbers of cells were given. Post-infusion laboratory investigations demonstrated an increase in the frequency of circulating anti-tumor T-cells and, in patients with a longer follow-up receiving two CTL infusions/year, a stabilization of these values.ConclusionsOur study demonstrates that autologous ex vivo-generated anti-tumor CTL can be administered safely in patients with advanced solid tumors and can improve the immunologic reactivity of recipients against tumor. These preliminary results provide a rationale for evaluating the clinical efficacy of this immunotherapeutic approach in phase I/II studies.     

Anti-asialo GM1 eliminates both inflammatory process and cytotoxic T-cell function in the lymphocytic choriomeningitis adoptive transfer model

The induction of severe inflammatory process and fatal neurological disease by transfer of lymphocytic choriomeningitis virus (LCMV)-immune T cells into cyclophosphamide (Cy)-suppressed LCMV-infected mice is greatly inhibited by treatment of these recipients with antibody to the asialo GM1 ganglioside (anti-ASGM1). Examination of cytotoxic activity in lymphoid tissue of the Cy-suppressed recipients at 72 hr after cell transfer revealed that anti-ASGM1 treatment prevented the development of the cytotoxic T lymphocyte (CTL) response, even though the dose of antibody used did not significantly decrease CTL generation in unsuppressed mice. Abrogation of CTL activity was also observed following antibody treatment of NK-deficient (bg/bg) Cy-suppressed recipients, indicating that the anti-ASGM1 was unlikely to be operating via removal of NK cells that are in some way involved in the development of the CTL response. The possibility that anti-ASGM1 may act directly on T cells should be considered in all protocols involving the use of this reagent in immunosuppressed mice.     

Immunopotentiation with low-dose cyclophosphamide in the active specific immunotherapy of cancer

This paper reviews the use of low-dose cyclophosphamide (CY) with active specific immunotherapy in patients with advanced melanoma and other metastatic cancers, and outlines the basic scientific research that supports this use. In various animal models, CY augments delayed-type hypersensitivity responses, increases antibody production, abrogates tolerance, and potentiates antitumor immunity. The mechanism of CY immunopotentiation involves inhibition of a suppressor function, as indicated by extensive work in the MOPC-315 plasmacytoma murine model. Human studies of the immunopotentiating effect of CY have yielded both positive and negative results. Toxicity associated with low-dose CY has been mild in these studies. Results of efficacy have been variable for reasons such as small sample sizes, short follow-up periods, and the weaker immunogenicity of human tumor-associated antigens. Although beneficial clinical outcomes have been observed in historically controlled trials, there are few randomized, controlled trials that evaluate outcome in relation to CY immunopotentiation of active specific immunotherapy. Additional randomized, controlled trials should be done to examine the clinical efficacy of CY immunopotentiation of therapeutic cancer vaccines. Received: 3 April 1998 / Accepted: 6 May 1998     

Active,specific immunotherapy of stage III breast cancer

Summary Active specific immunization with autologous irradiated tumor cells (AITC) admixed with BCG was attempted in 49 stage III breast cancer patients whose median follow-up at present is 3 years. As a first immunizing procedure 41 patients received repeated intradermal inoculations and 8 had a single endolymphatic instillation (ELI). Skin response (SR) to AITC alone was induced after two to nine weekly immunizations. Eight of 41 patients with negative or weak responses were effectively reimmunized by ELI, as indicated by conversion and invigoration of SR. Following immunization, radiotherapy to breast and axilla was administered. Thereafter, fortnightly 5-FU and monthly boosters of AITC-BCG mixture were given for 2 years. Strength of response to AITC induced by active immunization was found to relate to subsequent disease recurrence, with 15% relapsing among the good responders and 53% among the weak and non-responders. Positive SR to AITC — once elicited — was steadily maintained in the majority of patients; its decline was associated with manifest disease recurrence. Conversion to AITC positivity in vivo following specific immunization was not detectable by the LMI assay. Lymphocyte stimulation (MLTI) by AITC in vitro was found in only 9 of 26 tested patients with positive in vivo SR to AITC. Cutaneous response to AITC appears to be the only parameter of antitumor response showing clinical correlation, while specific in vitro correlates of cell-mediated immunity were found unsuitable for monitoring patients undergoing specific immunotherapy. While in vivo PPD response was mainly unchanged or enhanced, in vitro lymphocyte stimulation by PPD and PHA showed a distinct decline at the time of relapse. The cumulative proportion of relapse among the immunotherapy patients at 3 years was 32% with mortality of 12% (13 relapsed, 5 died), both being significantly lower than reported results in stage III breast cancer without immunotherapy. It is concluded that specific immunization with AITC is feasible in most breast cancer patients with loco-regionally advanced disease and that this intervention is conducive to favorable modification of the course of their disease.     

Effect of adoptive T-cell immunotherapy on immunological parameters and prognosis in patients with advanced pancreatic cancer

Background aimsImmunotherapy is effective for many types of cancer, but its benefits in advanced pancreatic cancer, which has a poor prognosis, are not well established. In this study, the authors examined the effects of adoptive T-cell immunotherapy (ATI) on immune cell profiles and prognosis in patients with unresectable advanced pancreatic cancer.MethodsSeventy-seven patients with unresectable advanced pancreatic cancer were treated with six cycles of αβ T cells alone or in combination with chemotherapy or chemoradiation. Immune cell profiles in peripheral blood samples obtained before and after treatment were comprehensively evaluated by flow cytometry. Furthermore, associations between changes in immune cell frequencies and prognosis were determined.ResultsATI prolonged survival to 18.7 months compared with previous estimates of 6.2–11.1 months for patients treated with chemotherapy alone. ATI decreased CD3+CD4+CD8? T cell frequency in peripheral blood and increased CD3+CD4?CD8+ T cell frequency. An increase in CD3+ T cells and CD3+TCRγδ? T cells in peripheral blood after treatment was associated with a good prognosis.ConclusionsATI altered the immune profile in peripheral blood, including CD3+CD4?CD8+ T cells, and improved prognosis in pancreatic cancer.     

Combination of active specific immunotherapy or adoptive antibody or lymphocyte immunotherapy with chemotherapy in the treatment of cancer

Successful treatment of cancer patients with a combination of monoclonal antibodies (mAb) and chemotherapeutic drugs has spawned various other forms of additional combination therapies, including vaccines or adoptive lymphocyte transfer combined with chemotherapeutics. These therapies were effective against established tumors in animal models and showed promising results in initial clinical trials in cancer patients, awaiting testing in larger randomized controlled studies. Although combination between immunotherapy and chemotherapy has long been viewed as incompatible as chemotherapy, especially in high doses meant to increase anti-tumor efficacy, has induced immunosuppression, various mechanisms may explain the reported synergistic effects of the two types of therapies. Thus direct effects of chemotherapy on tumor or host environment, such as induction of tumor cell death, elimination of regulatory T cells, and/or enhancement of tumor cell sensitivity to lysis by CTL may account for enhancement of immunotherapy by chemotherapy. Furthermore, induction of lymphopenia by chemotherapy has increased the efficacy of adoptive lymphocyte transfer in cancer patients. On the other hand, immunotherapy may directly modulate the tumor’s sensitivity to chemotherapy. Thus, anti-tumor mAb can increase the sensitivity of tumor cells to chemotherapeutic drugs and patients treated first with immunotherapy followed by chemotherapy showed higher clinical response rates than patients that had received chemotherapy alone. In conclusion, combination of active specific immunotherapy or adoptive mAb or lymphocyte immunotherapy with chemotherapy has great potential for the treatment of cancer patients which needs to be confirmed in larger controlled and randomized Phase III trials.     

Active specific immunotherapy of guinea pigs with visceral tumor implants

Summary Guinea pigs, each with established, 7-day-old, syngeneic visceral micrometastases of line 10 tumor implanted intravenously, were immunized by intradermal inoculations into several sites of a mixture of irradiated line 10 cells and an emulsion containing heat-killed BCG or Mycobacterium phlei bacilli. This treatment led to survival of 72 of 80 treated animals (90%). Therapeutic effectiveness depended on the dose of mycobacteria and on that of irradiated tumor cells. Animals treated by intradermal injection of mycobacteria attached to oil droplets alone or with irradiated tumor cells alone, all died with multiple foci of pulmonary tumor.     

Restoration of immunity in lymphopenic individuals with cancer by vaccination and adoptive T-cell transfer

Immunodeficiency is a barrier to successful vaccination in individuals with cancer and chronic infection. We performed a randomized phase 1/2 study in lymphopenic individuals after high-dose chemotherapy and autologous hematopoietic stem cell transplantation for myeloma. Combination immunotherapy consisting of a single early post-transplant infusion of in vivo vaccine-primed and ex vivo costimulated autologous T cells followed by post-transplant booster immunizations improved the severe immunodeficiency associated with high-dose chemotherapy and led to the induction of clinically relevant immunity in adults within a month after transplantation. Immune assays showed accelerated restoration of CD4 T-cell numbers and function. Early T-cell infusions also resulted in significantly improved T-cell proliferation in response to antigens that were not contained in the vaccine, as assessed by responses to staphylococcal enterotoxin B and cytomegalovirus antigens (P < 0.05). In the setting of lymphopenia, combined vaccine therapy and adoptive T-cell transfer fosters the development of enhanced memory T-cell responses.     

Failure of specific adoptive immunotherapy owing to survival and outgrowth of variant cells

Summary Adoptive immunotherapy, the transfer of spleen cells from immunized mice to mice with a small tumor, was usually curative for mice with the P815 mastocytoma provided that steps were taken to prevent the generation of tumor-induced suppressor cells in the recipient animal. However, failure of adoptive immunotherapy of the P815 tumor, resulting in regrowth of either the primary intradermal or a metastatic tumor, was observed in 10 out of 112 animals receiving graded doses of 7.5×10⁷ to 3.0×10⁸ immune spleen cells. Examination of the ten tumors in mice that failed to respond to therapy revealed that seven of them were significantly less susceptible than the original P815 tumor to rejection in vivo by transferred anti-P815-specific effector cells. In addition, nine of the ten therapy-failure tumors were also less susceptible than the original P815 tumor to lysis in vitro by P815-specific, but not DBA/2-specific, cytotoxic T lymphocytes. Sensitivity to lysis by tumor-specific cytotoxic T cells was not, however, strongly correlated with sensitivity to rejection in vivo by P815-specific effector spleen cells. Neither in vivo sensitivity to rejection, nor sensitivity to cytotoxic T cells, was correlated with alterations in class I major histocompatibility complex antigen expression. These results suggest that the survival and outgrowth of variant tumor cells was frequently the cause of failure of specific adoptive immunotherapy of the P815 tumor, and that selection for cells with a reduced sensitivity to killing by cytotoxic T cells was only one mechanism that might lead to an immunotherapeutic failure.This work was supported by a grant from RJR Nabisco Inc., a grant from the J. M. Foundation, and by USPHS grant CA-40597 awarded by the National Cancer Institute     

Human T-cell leukemia virus type I infection of CD4+ or CD8+ cytotoxic T-cell clones results in immortalization with retention of antigen specificity.

The human T-cell leukemia virus type I (HTLV-I) is capable of chronically infecting various types of T cells and nonlymphoid cells. The effects of chronic infection on the specific functional activities and growth requirements of mature cytotoxic T lymphocytes (CTL) have remained poorly defined. We have, therefore, investigated the results of HTLV-I infection of both CD4+ and CD8+ human CTL clones. HTLV-I infection resulted in the establishment of functional CTL lines which propagated indefinitely in culture many months longer than the uninfected parental clone. The infected cells became independent of the need for antigen (target cell) stimulation as a requirement for proliferation and growth. Like their uninfected counterparts, however, these HTLV-I-infected clones remained strictly dependent on conditioned medium from mitogen-stimulated T lymphocytes for their growth. This growth factor requirement was not fulfilled by recombinant interleukin-2 alone. Furthermore, the infected lines remained functionally identical to their uninfected parental CTL clones in their ability to specifically recognize and lyse the appropriate target cells. Our findings indicate that the major effects of HTLV-I infection on mature CTL consist of (i) the capacity for proliferation in the absence of antigen stimulation and (ii) a prolonged or immortal survival in vitro, but they also indicate that the fine specificity and cytolytic capacity of these cells remain unaffected.     

Theileria parva: CD4+ helper and cytotoxic T-cell clones react with a schizont-derived antigen associated with the surface of Theileria parva-infected lymphocytes.

Theileria parva is a protozoan parasite which infects and transforms bovine lymphocytes, resulting in a fatal lymphoproliferative disease. There is evidence that immunity to the intralymphocytic schizont stage is mediated by T cells. We have previously reported derivation of CD4+ T-cell clones which recognize parasite-derived antigens presented on the surface of infected cells in conjunction with MHC molecules and partial characterization of the antigens. The present study further evaluated one of these antigens, demonstrating that it could be derived from cells infected with different parasite stocks as well as from purified theilerial schizonts and that it was recognized by primed, but not unprimed, bovine lymphocytes including cytolytic CD4+ T cells. Using a cloned CD4+ cytolytic cell line, lysis of schizont-infected cells was shown to be MHC-restricted but not parasite-strain restricted. In addition we demonstrated that T cells which respond to the HSS antigen preparation were generated in cattle immunized with parasites from any of the three subspecies of T. parva. The antigenic material was fractionated by sequential subjection to anion-exchange chromatography, hydroxylapatite chromatography, and gel filtration using HPLC, which resulted in recovery of approximately 20% of the antigenic material with more than 10(6)-fold purification in selected fractions. To assess the molecular size of the proteins in the highly purified antigenic fractions, the T. parva-infected lymphocytes were metabolically labeled before fractionation with 3H-amino acids and the material was analyzed by SDS-polyacrylamide gel electrophoresis and liquid scintillation counting of gel slices. The major protein in these fractions had a molecular mass of 9-10 kDa.     

Demonstration of multiple antigenic sites of the SV40 transplantation rejection antigen by using cytotoxic T lymphocyte clones

Multiple antigenic sites on the simian virus 40 (SV40) tumor-specific transplantation antigen (TSTA) were detected by the use of cytotoxic T lymphocyte (CTL) clones isolated from continuous cultures of SV40-specific CTL (H-2b). Two independently derived clones, K11 and K19, specific for the SV40 TSTA in association with H-2Db, each recognized a different antigenic determinant of the SV40 TSTA. This conclusion was based on the observation that a human papovavirus BK virus (BKV) transformed cell line, which possesses a T antigen serologically cross-reactive with that of SV40, was lysed by a heterogeneous population of SV40-immune lymphocytes and by clone K19 but not by K11. Therefore, these CTL clones must recognize two different antigenic determinants of the SV40 TSTA:K19 recognizes a cross-reactive determinant of the SV40 and BKV TSTA, whereas K11 is reactive against an SV40-specific determinant.     

Lymphadenectomy exacerbates tumor growth while lymphadenectomy plus the adoptive transfer of autologous cytotoxic cells and low-dose cyclophosphamide induces regression of an established murine fibrosarcoma

Tumor-draining lymph node (TDLN) ablation is routinely performed in the management of cancer; nevertheless, its usefulness is at present a matter of debate. TDLN are central sites where T cell priming to tumor antigens and onset of the antitumor immune response occur. However, tumor-induced immunosuppression has been demonstrated at TDLN, leading to downregulation of antitumor reaction and tolerance induction. Tolerance in turn is a main impairment for immunotherapy trials. We used a murine immunogenic fibrosarcoma that evolves to a tolerogenic state, to study the cellular and molecular mechanisms underlying tolerance induction at the level of TDLN and to design an appropriate immunotherapy. We determined that following a transient activation, the established tumor induces signs of immunosuppression at TDLN that coexist with local and systemic evidences of antitumor response. Therefore, we evaluated the feasibility of removing TDLN in order to eliminate a focus of immunosuppression and favor tumor rejection; but instead, a marked exacerbation of tumor growth was induced. Combining TDLN ablation with the in vivo depletion of regulatory cells by low-dose cyclophosphamide and the restoring of the TDLN-derived cells into the donor mouse by adoptive transference, resulted in lowered tumor growth, enhanced survival and a considerable degree of tumor regression. Our results demonstrate that important antitumor elements can be eliminated by lymphadenectomy and proved that the concurrent administration of low-dose chemotherapy along with the reinoculation of autologous cytotoxic cells provides protection. We suggest that this protocol may be useful, especially in the cases where lymphadenectomy is mandatory.