Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells.

Intratumoral implantation of murine cells modified to produce retroviral vectors containing the herpes simplex virus-thymidine kinase (HSV-TK) gene induces regression of experimental brain tumors in rodents after ganciclovir (GCV) administration. We evaluated this approach in 15 patients with progressive growth of recurrent malignant brain tumors. Antitumor activity was detected in five of the smaller tumors (1.4 +/- 0.5 ml). In situ hybridization for HSV-TK demonstrated survival of vector-producing cells (VPCs) at 7 days but indicated limited gene transfer to tumors, suggesting that indirect, "bystander," mechanisms provide local antitumor activity in human tumors. However, the response of only very small tumors in which a high density of vector-producing cells had been placed suggests that techniques to improve delivery and distribution of the therapeutic gene will need to be developed if clinical utility is to be achieved with this approach.

T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates.

Adenosine deaminase-deficient severe combined immunodeficiency was the first disease investigated for gene therapy because of a postulated production or survival advantage for gene-corrected T lymphocytes, which may overcome inefficient gene transfer. Four years after three newborns with this disease were given infusions of transduced autologous umbilical cord blood CD34+ cells, the frequency of gene-containing T lymphocytes has risen to 1-10%, whereas the frequencies of other hematopoietic and lymphoid cells containing the gene remain at 0.01-0.1%. Cessation of polyethylene glycol-conjugated adenosine deaminase enzyme replacement in one subject led to a decline in immune function, despite the persistence of gene-containing T lymphocytes. Thus, despite the long-term engraftment of transduced stem cells and selective accumulation of gene-containing T lymphocytes, improved gene transfer and expression will be needed to attain a therapeutic effect.

Peripheral expansion of pre-existing mature T cells is an important means of CD4+ T-cell regeneration HIV-infected adults.

The CD4+ T-cell pool in HIV-infected patients is in a constant state of flux as CD4+ T cells are infected and destroyed by HIV and new cells take their place. To study T-cell survival, we adoptively transferred peripheral blood lymphocytes transduced with the neomycin phosphotransferase gene between syngeneic twin pairs discordant for HIV infection. A stable fraction of marked CD4+ T cells persisted in the circulation for four to eighteen weeks after transfer in all patients. After this time there was a precipitous decline in marked cells in three of the patients. At approximately six months, marked cells were in lymphoid tissues in proportions comparable to those found in peripheral blood. In two patients, the proportion of total signal for the transgene (found by PCR analysis) in the CD4/CD45RA+ T-cell population relative to the CD4/CD45RO+ population increased in the weeks after cell infusion. These findings indicate that genetically-marked CD4+ T cells persist in vivo for weeks to months and that the CD4+ T-cell pool in adults is maintained mostly by the division of mature T cells rather than by differentiation of prethymic stem cells. Thus, after elements of the T-cell repertoire are lost through HIV infection, they may be difficult to replace.

T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years.

In 1990, a clinical trial was started using retroviral-mediated transfer of the adenosine deaminase (ADA) gene into the T cells of two children with severe combined immunodeficiency (ADA- SCID). The number of blood T cells normalized as did many cellular and humoral immune responses. Gene treatment ended after 2 years, but integrated vector and ADA gene expression in T cells persisted. Although many components remain to be perfected, it is concluded here that gene therapy can be a safe and effective addition to treatment for some patients with this severe immunodeficiency disease.

Morphology of interleukin-2-stimulated human peripheral blood mononuclear effector cells killing glioma-derived tumor cells in vitro.

This is the first morphological study of interleukin-2-stimulated human peripheral blood mononuclear (PBM) cells resulting in lymphokine-activated killer (LAK) cell activity against human glioma-derived tumor cells in vitro, in which high-resolution differential interference video light microscopy, scanning electron microscopy, and transmission electron microscopy were used. A subset of cells within the LAK cell population are the effector cells and have an asymmetric cellular architecture characteristic of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Upon binding to target cells, the LAK effector cell nucleus is positioned away from the target cell, whereas the granules, Golgi apparatus, and microtubules orient toward the target cell. These LAK-glioma cell conjugates form very tight plasma membrane bonds with numerous interdigitations, and vesicles were found in the small extracellular spaces between the cells. This morphology was not observed in unstimulated PBM-glioma cell co-cultures. Glioma-derived cells react to LAK effector cells by blebbing, becoming round, and rapidly detaching from the substrate. The injured glioma-derived cells had a highly condensed cytoplasm and chromatin, lobular nucleus, and severe plasma membrane blebs, which are consistent with an apoptotic rather than an osmotic lysis mechanism of cell death. This study provides morphological evidence that supports a common cytotoxic mechanism for CTLs, NK cells, and LAK effector cells. The cytotoxic mechanism is based on the local exocytosis of vesicles by the effector cell into the small extracellular space between the effector-target cell conjugate. Granules found in CTLs, NK cells, and LAK cells contain a pore-forming protein that inserts holes in the target cell's plasma membrane through which a lethal substance(s) not yet identified is thought to enter the cell.

Interleukin 2 expanded tumor-infiltrating lymphocytes in human renal cell cancer: isolation, characterization, and antitumor activity.

We have described a method for the generation, from fresh human renal cell cancers, of lymphoid cells that are capable of exhibiting significant antitumor reactivity when tested in short term 51Cr release assays. Tumor cell suspensions obtained from 37 consecutive fresh human renal cell cancer specimens (35 patients) could be separated by using enzymatic techniques and culturing in medium containing recombinant interleukin 2 (IL-2). The total cell recovery was 1.5 X 10(9) +/- 2.2 (SE) per tumor with a range of 1 X 10(8) to 5 X 10(9) cells. The percentage of tumor cells in the suspension ranged from 6 to 75% with a mean of 39.1 +/- 3.3%. The remaining cells were predominantly lymphocytes. Viability of mononuclear cells was greater than 90%. Activated tumor-infiltrating lymphocytes (TIL) within these tumors expand and by 10 to 14 days after initiation of culture a 5- to 15-fold increase in the number of lymphocytes could be achieved with elimination of all autologous tumor cells. Lymphocytes were recultured in fresh medium containing IL-2 and continued to expand between 2- and 10-fold every 4 to 6 days for an average of 33.7 +/- 4.5 days, resulting in greater than 50,000-fold increase in the total number of lymphocytes. The average number of splits was 4.9 +/- 0.8, with a range of 0 to 21. In 11 of 11 cases tested, TIL exhibited a far better expansion capability in vitro compared to that of peripheral blood lymphocytes obtained from the same patient and grown under identical conditions. The majority of TIL were T cytotoxic/suppressor cells (Leu 2+ Leu 4+). With continued in vitro expansion (up to 50 days) there was a concomitant increase in the helper T (Leu 3+) and pan T populations (Leu 4+) and decrease in Leu 2+ and HLA-DR+ cells. Compared with expanded peripheral blood lymphocytes, these cells demonstrated higher levels of IL-2+ receptors and HLA-DR+ antigens. Renal TIL effectors expanded in IL-2 could lyse almost all autologous tumor targets in 4-h chromium release assays. Allogeneic renal as well as nonrenal targets were equally lysed. TIL lysis of cultured tumor targets K562 and Daudi was significantly better than lysis of autologous, allogeneic-renal, and nonrenal targets. No statistically significant difference in the cytotoxic activity of renal TIL or peripheral blood lymphocyte effectors in killing autologous or allogeneic targets could be demonstrated.(ABSTRACT TRUNCATED AT 400 WORDS)

Neoplasms of immunoregulatory T cells in clinical investigation.

Normal T cells play a critical role in the regulation of humoral immune responses by acting as potentiators (helper cells) or inhibitors (suppressor cells) of the process by which B cells differentiate into immunoglobulin-secreting plasma cells. Certain diseases in which malignant T cells appear to retain an immunoregulatory function are characterized by a propensity of a lymphomatous T-cell population to infiltrate skin. Some cutaneous T-cell lymphomas, as well as some T-cell neoplasms without dermatologic involvement, provide a homogeneous supply of T lymphocytes which act as immunoregulators. The availability of neoplastic T cells with immunoregulatory properties could accelerate the serologic and biochemical analysis of the cellular control of normal immunity in man.

Expansion of human tumor infiltrating lymphocytes for use in immunotherapy trials.

The potential utility of tumor-infiltrating lymphocytes (TIL) in the adoptive immunotherapy of human tumors has been suggested by murine experiments showing these cells to be 50-100 times more powerful than LAK cells in treating advanced metastatic disease. A method for the large-scale expansion of human TIL for the use of these cells in clinical trials is described in this report. TIL were successfully expanded on an experimental scale from 24 of 25 consecutive human tumors, including six melanomas, ten sarcomas, and eight adenocarcinomas. Tumors were digested enzymatically to yield single cell suspensions which were cultured in RPMI 1640 medium with 10% human serum and 1000 U/ml recombinant interleukin-2. Lymphocytes constituted from 3% to 74% of single cell tumor suspensions, and expanded from 2.9-fold to 9.1 X 10(8)-fold over a culture period ranging from 14 to 100 days. Nine of 24 TIL cultures lysed fresh autologous tumor targets in 4 h chromium release assays. Cell surface phenotyping identified cultured TIL as activated cytotoxic/suppressor T cells. Subsequently, large-scale expansion of TIL was successful in generating more than 10(10) lymphocytes in five of eight consecutive cases. Clinical trials employing the adoptive transfer of expanded TIL to patients with metastatic disease have begun.

Large scale production of human lymphokine activated killer cells for use in adoptive immunotherapy.

Immunotherapy utilizing the adoptive transfer of lymphokine activated killer (LAK) cells in conjunction with recombinant interleukin 2 (RIL-2) is capable of reducing established metastatic cancer in a variety of animal tumor models. A major difficulty in the application of these efforts to the treatment of human cancer has been the activation in vitro of up to 2 X 10(11) human peripheral blood lymphocytes obtained by repeated leukaphereses. We have thus developed optimal and simplified techniques for the generation of human LAK cells for use in clinical trials. We have found that 1.5 X 10(9) lymphocytes separated on Ficoll-Hypaque gradients and incubated in 1000 ml of culture medium in a 2.3 liter roller bottle with 1000-1500 U of RIL-2 per ml, generated LAK cells capable of killing fresh human tumor cells in a 4 h chromium release assay. The culture medium used was RPMI 1640 with 2 mM glutamine, 2% heat-inactivated human AB serum, 50 micrograms/ml streptomycin and gentamicin and 50 U/ml penicillin. This technique allows activation of sufficient numbers of cells in a research laboratory setting to conduct human clinical trials. The administration of LAK cells generated in this fashion can mediate the regression of human tumors when administered in conjunction with IL-2.

Studies of serum-free culture medium in the generation of lymphokine activated killer cells.

Lymphokine activated killer (LAK) cells administered in conjunction with recombinant interleukin-2 can mediate the regression of metastatic tumor in some patients with advanced cancer. In these trials LAK cells were activated in medium containing 2% human type A or AB serum. We have found three commercially available, serum-free culture media which allow development of in vitro LAK activity by human peripheral blood lymphocytes. They are AIMV (Gibco), MASF-3 (Whitaker-MA Bioproducts) and HB-104 (Dupont). If 2-mercaptoethanol was added to these culture media they were also capable of generating murine LAK cells which were effective in reducing pulmonary metastases in the murine MCA-106 model. Although LAK cells generated in these media have not been tested in humans yet, potentially they could provide a safe, unlimited and less expensive source of culture fluid for generating the large numbers of LAK cells needed for human clinical trials.

Development of an automated closed system for generation of human lymphokine-activated killer (LAK) cells for use in adoptive immunotherapy.

Immunotherapy utilizing the adoptive transfer of lymphokine-activated killer (LAK) cells in conjunction with recombinant interleukin-2 (IL-2) can mediate tumor regression in some patients with advanced cancer. The activation of large numbers of LAK cells was performed in roller bottles in a research laboratory setting and required meticulous aseptic technique, at least one skilled technician per patient and one laminar flow hood per patient. To reduce the complexity and expense of LAK cell generation for human immunotherapy trials we have developed a closed-system automated procedure using a continuous flow blood cell separator. PBL were obtained by standard apheresis techniques. Platelets and plasma were elutriated using countercentrifugal flow of saline in the cell separator machine. The washed PBL were underlaid with Ficoll-Hypaque (FH) in the original separation bag. Lymphocytes were then flushed into a collection bag where they were concentrated and washed with 2 liters of saline. Mean recovery from the automated FH technique was 54.6 +/- 4.3% compared to 62.3 +/- 4.0% using manual methods in 50 ml tubes (P greater than 0.05). Cells were diluted in the collection bag with RPMI 1640 +/- 2% human AB serum and could be dispensed in an automated fashion to polyolefin bags via a sample port with 1000-1500 U/ml IL-2. After 3-4 days of culture in 5% CO2 at 37 degrees C, activated cells from the bags were harvested and washed in a closed system using the continuous flow cell separator. Cell yield from the harvest was 79.2 +/- 5.4% in the automated system compared to 64.9 +/- 5.0% in the standard procedure using manual harvest of roller bottles (P less than 0.01). Lytic capacity of the cells against fresh human tumor in a 4 h 51Cr release assay was equivalent in cells processed either by the automated or the conventional manual method. The advantages of a closed system include decreased potential for microbial contamination and reduced labor and capital equipment costs. This technique may be easily adapted for use with other cell collection and culture systems.

The differential inhibitory effects exerted by cyclosporine and hydrocortisone on the activation of human cytotoxic lymphocytes by recombinant interleukin-2 versus allospecific CTL.

Two immunosuppressive drugs, cyclosporine (CsA) and Hydrocortisone (Hy) were examined in parallel for their effect on the generation of cytolytic T lymphocytes (CTL). Peripheral blood lymphocytes (PBL) were stimulated with allogeneic cells to produce allospecific CTL, or with purified recombinant Interleukin 2 to activate lymphokine activated killer cells (LAK). CTL and LAK activity were measured in a 4 hr chromium release assay after 7 days of activation. Lysis by CTL was tested against stimulator PBL (not blasts) and LAK against fresh sarcoma tumor cells. At pharmacologic doses, CsA inhibited only CTL generation, and Hy inhibited only LAK. This greater understanding of the selective role, or roles, in vitro of CsA and Hy provides a basis by which to consider selective immune suppression--and, alternatively, the possibility of combining modalities for a more thorough immune suppression.

A new approach to the therapy of cancer based on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2.

A new approach to cancer therapy has been developed based on the adoptive transfer of autologous lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (IL-2). Forty-one patients with advanced cancer who have failed all standard treatments were treated in this experimental protocol. Fourteen patients experienced an objective regression of cancer, including one patient with metastatic melanoma who underwent a complete regression. Objective responses were seen in patients with colorectal cancer, renal cell cancer, melanoma, and lung adenocarcinoma. The sites of tumor regression included subcutaneous tissue, lung, and liver. The major side effect of therapy resulted from the administration of high-dose IL-2 and was manifested primarily as fluid retention, resulting in a generalized capillary permeability leak syndrome. This approach to adoptive immunotherapy represents a promising approach to the therapy of patients with metastatic cancer. Attempts to increase the potency and decrease the toxicity of therapy and extend this treatment to patients with smaller tumor burdens are in progress.

Hydrocortisone suppresses the generation of nonspecific "anomalous" killers but not specific cytolytic T lymphocytes in human mixed lymphocyte-tumor cultures.

Interpretation of results with the use of effector populations generated in human mixed lymphocyte tumor cultures (MLTC) has in many cases been clouded by the presence of two distinct cytolytic effectors: specific cytolytic T lymphocytes (CTL) and nonspecific anomalous killers (AK). The addition of 5 to 50 X 10(-6) M hydrocortisone sodium succinate (HC) to "three-cell" allogeneic human MLTC containing responder lymphocytes, irradiated allogeneic glioma cells, and irradiated third-party stimulator lymphocytes consistently reduced the generation of AK by greater than 90% while having little or no effect on the generation of CTL specific for antigens present on the glioma cells. HC was similarly effective in blocking the generation of AK when added to cultures of responder cells incubated with lymphokine-containing supernatants from mixed lymphocyte cultures. Kinetic experiments indicated that HC both decreased the initial rate of appearance of AK activity and subsequently caused the AK response to decline more rapidly. HC had only a small inhibitory effect on the lytic action of AK generated in its absence. The ability of HC to suppress strongly the generation of nonspecific but not specific cytolytic effectors suggests that nonspecific AK are not polyclonally activated CTL or CTL at an early stage of differentiation, but rather represent a distinct cytolytic effector system. The inclusion of HC in human MLTC may provide a useful tool for distinguishing between lysis due to the generation of specific CTL and lysis due to the generation of nonspecific AK.

Polyclonal activation of the murine immune system by an antibody to IgD. III. Ontogeny.

It has recently been demonstrated that the injection of adult mice with an affinity-purified goat antibody to mouse IgD (GaM delta) stimulates activation of the humoral immune system that resembles, on a polyclonal level, specific B cell activation by a T cell-dependent antigen. One to 2 days after adult BALB/c mice are injected with 200 micrograms of GaM delta, their splenic B lymphocytes undergo a series of T-independent activation steps that include increases in surface (s) Ia expression, cell size and DNA synthesis. Seven days after GaM delta injection, these cells undergo T-dependent activation steps, that include further proliferation as well as differentiation into IgG1-secreting cells. We have now studied the ontogeny of the T-independent (day 2) and T-dependent (day 7) activation steps by injecting 100-200 micrograms of GaM delta into 3-day- to 10-week-old BALB/c mice. GaM delta failed to induce increases in B cell sIa expression or size 2 days after injection of mice 2 weeks old or younger and failed to stimulate increased DNA synthesis 2 days after injection of 4-week-old mice. In contrast, increases in spleen cell sIa expression, size and DNA synthesis were seen 7 days after injection of 6- to 8-day-old mice. Furthermore, increases in the numbers of spleen cells with large amounts of intracytoplasmic IgG1 were seen at the same time, although these increases were much less than were seen in GaM delta-treated adult mice. Thus, the ability of GaM delta to induce T help and to act in concert with such help to stimulate B cell proliferation and differentiation precedes in ontogeny the ability of GaM delta to directly induce B cell proliferation and early differentiative events. In addition, the early activating events that we have studied are not required for T-dependent B cell proliferation and antibody production to occur, although they appear to contribute to the magnitude of clonal expansion and antibody production.

Interactions between radioresistant T cells from plasmacytoma-bearing mice and radiosensitive T cells from normal mice leading to suppression.

Spleens from BALB/c mice bearing the plasmacytoma SPQC 11 contain a population of T cells capable of suppressing the production of antibodies when cultured with normal BALB/c splenocytes, but not with splenocytes from nude BALB/c mice. Inhibition was characteristically the most effective at low suppressor cell numbers using unirradiated T cells. Maximum suppression occurred when a radiosensitive population of T cells from normal mice interacted with a population of radioresistant T cells from plasmacytoma-bearing animals. Accurate characterization of the suppressor T cell activity required testing over a wide range of suppressor/indicator cell ratios. Suppressor activity was eliminated by treatment with anti-Thy 1.2 plus complement, was radioresistant and present over a full range of doses when irradiated cells were used.

Polyclonal activation of the murine immune system by an antibody to IgD. IV. In vivo activation of B lymphocytes from immune defective CBA/N mice.

Previous studies with BALB/c mice have established that the injection of a goat antibody to IgD (GaM delta) induces splenic B lymphocytes to undergo T-independent, polyclonal increases in cell surface (s) Ia expression, size, and DNA synthesis 1 day after injection, as well as T-dependent maintenance of this activation and differentiation into sIgG1+ cells and IgG1-secreting cells 6 to 7 days after injection. Because B lymphocytes from mice homozygous or hemizygous for the CBA/N X-linked immune defect fail to proliferate in vitro when cultured with soluble anti-Ig antibodies, yet can make in vivo primary antibody responses to T-dependent antigens, we studied the effects of injecting (CBA/N X DBA/2)F1 male mice (immune defective) and (CBA/N X DBA/2)F1 female mice (phenotypically normal) with 800 to 1600 micrograms of GaM delta. B cells from immune defective mice demonstrated definite but distinctly subnormal increases in B cell size, DNA synthesis, and sIa expression 1 to 5 days after GaM delta injection, but by day 7 showed increases in these parameters as well as in the percentages of splenic sIgG1+ cells and IgM-secreting cells that were similar to those exhibited by the immunologically normal mice. However, IgG1 secretion by the immune defective mice was at this time only one-fourth to one-half as great as that observed with normal mice. Our data raises the possibility that the differentiation of B cells into IgG1-secreting cells may be more dependent upon the GaM delta-induced activation steps that are defective in CBA/N mice than is the differentiation of B cells into IgM-secreting cells or into sIgG1 cells.

Identification of specific cytolytic immune responses against autologous tumor in humans bearing malignant melanoma.

Tumor-infiltrating lymphocytes from six patients with metastatic malignant melanoma were expanded by culture in recombinant interleukin 2. Three of the preparations were highly cytotoxic against autologous fresh melanoma tumor cells, but not against autologous fresh normal cells or allogeneic fresh tumor targets. The other three were highly cytotoxic against autologous fresh melanoma tumor cells and also had a limited capacity to kill allogeneic fresh tumor targets. The tumor-associated specific killer cells could be expanded from threefold to 95,652-fold with maintenance of specific antitumor lysis. The expanded tumor-infiltrating cells were Leu-4+ T cells, and in five of six patients the majority were Leu-3+. These studies demonstrate that the melanoma-bearing patient raises an immune response against autologous tumor and presents a method for the generation of human lymphocytes with antitumor reactivity that may be useful in the adoptive immunotherapy of tumors.