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Background Aims: Several studies have demonstrated that the immunogenicity of chronic lymphocytic leukemia (CLL) cells can be increased by manipulation of the CD40/CD40-ligand (CD40L) pathway. Although immunologic, and perhaps clinical, benefits have been obtained with an autologous CLL tumor vaccine obtained by transgenic expression of CD40L and interleukin (IL)-2, there is little information about the optimal gene transfer strategies., Methods: We compared two different CLL vaccines prepared by adenoviral gene transfer and plasmid electroporation, analyzing their phenotype and immunostimulatory activity., Results: We found that higher expression of transgenic CD40L was mediated by adenoviral gene transfer than by plasmid transduction, and that adenoviral transfer of CD40L was associated with up-regulation of the co-stimulatory molecules CD80 and CD86 and adhesion molecule CD54. In contrast, transgenic IL-2 secretion was greater following plasmid transduction. These phenotypic differences in the vaccines were associated with different functionality, both ex vivo and following administration to patients. Thus adenoviral vaccines induced greater activation of leukemia-reactive T cells ex vivo than plasmid vaccines. In treated patients, specific T-cell (T helper 1 (Th1) and T helper 2 (Th2)) and humoral anti-leukemia responses were detected following administration of the adenoviral vaccine (n = 15), while recipients of the plasmid vaccine (n = 9) manifested only a low-level Th2 response. Progression-free survival at 2 years was 46.7% in the adenoviral vaccine recipients, versus 11.1 % in those receiving plasmid vaccine., Conclusions: CLL vaccines expressing the same transgenes but produced by distinct methods of gene transfer may differ in the polarity of the immune response they induce in patients.

Although IL-2 infusion enhances cell-mediated cytotoxicity in patients with neoplastic disease, administration is paradoxically associated with a modest fall in total serum IgG and an increased risk of infection. We now show that the adverse effects of IL-2 infusion on the humoral immune system are substantial. Although IL-2 induces the B cell growth and differentiating factors IL-4 and IL-6, infusion abrogates primary antibody responses entirely and reduces secondary antibody responses 50-fold following antigen challenge. There is no evidence of the generation of cells with suppressive activity on B cells but IL-2 increases the ratio of circulating virgin:memory cells. These results may help to explain the increased rate of bacterial infection in patients receiving IL-2. As IL-2 plays a central role in the generation of an immune response, the finding that it is also sufficiently immunosuppressive to inhibit primary- and secondary-type antibody responses suggests that exploration of the underlying mechanisms may provide insights into immune system homeostasis and may offer new approaches to therapeutic immunosuppression.

Cytokine-secreting, major histocompatibility complex-unrestricted activated killer (AK) cells are toxic to a wide range of virus-infected or malignant target cells and may be generated endogenously, eg, after bone marrow transplantation, or by infusion of cytokines such as recombinant interleukin-2 (rIL-2). Although AK cells secrete cytokines such as gamma-interferon and tumor necrosis factor, which are themselves able to recruit fresh cytokine-secreting AK cells, activation in both settings is short-lived, implying the existence of homeostatic regulatory mechanisms. We now demonstrate one mechanism by which rapid homeostasis is achieved. We show that IL-4 is produced in patients with both endogenously and exogenously generated AK cells. The cytokine was detected in serum after marrow transplantation, and IL-4 transcripts appeared in circulating lymphocytes during rIL-2 infusion. Although IL-4 inhibited the induction phase of AK cell function, it had no significant inhibitory effect on the ability of AK cells from these individuals to respond to restimulation. Nonetheless, neutralization of the IL-4 induced during cell activation doubled the half-life of AK function, once activating stimuli were removed, from 18 to 44 hours and produced a 2-log increase in AK cell secretion of tumor necrosis factor and gamma-interferon. These data suggest that IL-4 induced in vivo during lymphocyte activation abbreviates AK cell responses once the triggering stimuli have been removed. Neutralization of endogenous IL-4 in vivo by appropriate monoclonal antibodies might prolong the duration of AK function.

Administration of interleukin 2 (IL2) to patients with minimal residual malignant disease following myeloablative chemo-radiotherapy may augment immune reconstitution and reduce the risk of relapse by increasing cytotoxic effector function and cytokine dependent killing directed at residual malignant cells. The ability of IL2 generated activated killer cells to inhibit haemopoietic progenitor cells and to release gamma-interferon (gamma IFN) and tumour necrosis factor (TNF) may, however, retard haemopoietic recovery, as both TNF and gamma IFN inhibit normal myelopoiesis in vitro. To determine the effect of IL2 infusion on myeloid regeneration in vivo, we have examined haemopoietic recovery in patients receiving this cytokine following autologous marrow transplantation or ablative chemotherapy. We find that IL2 infusion accelerates neutrophil recovery and that granulocyte-macrophage colony stimulating factor (GMCSF) and IL3 mRNA become detectable in circulating mononuclear cells. Induction of TNF by IL2 may also contribute to subsequent acceleration of myelopoiesis by initiation of GM-CSF mRNA synthesis in patient marrow fibroblasts. These results show that IL2 infusion may facilitate myeloid recovery when administered during the period of haemopoietic regeneration following ablative chemoradiotherapy.

To assess the role of the cytokine interleukin 2 (IL2) in the treatment of patients with multiple myeloma, we examined the sensitivity of plasma cell lines and malignant plasma cells from multiple myeloma (MM) patients to cell and cytokine-mediated killing induced by IL2. Unstimulated peripheral blood mononuclear cells (PBM) from normal donors produced little killing (mean lysis 1.0 +/- 1.0%, effector:target (ET) ratio 50:1), but cytotoxicity was modestly increased when PBM were incubated with IL2 prior to assay (8.0 +/- 2.9%). Unstimulated PBM from patients with MM also failed to kill autologous malignant plasma cells (mean 0.6 +/- 0.6%), but after exposure to IL2 they induced substantial lysis of autologous malignant cells (mean 55.3 +/- 22.1%). In addition, tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma), two cytokines released from mononuclear cells in response to IL2, also reduced the survival and thymidine uptake of malignant plasma cells in culture. To determine whether these potentially beneficial immunomodulatory effects could be reproduced by in vivo administration of IL2, we have administered seven courses of IL2 to four patients with MM after autologous bone marrow transplant (ABMT). No serious adverse effects were noted. Increases in natural killer (NK) and lymphokine-activated killer (LAK) activity of PBM occurred during IL2 infusion, although cells capable of killing autologous MM cells did not circulate. However, IL2 infusions also induced substantial increases in the production of the cytokines TNF and IFN-gamma from peripheral blood lymphocytes. These results suggest that the in vivo administration of IL2 in MM deserves further evaluation, particularly for its potential to control minimal residual disease.

Biological response modifiers such as interleukin 2 (IL2) may be most effective in the setting of minimal residual disease. In a phase I-II clinical trial, IL2 was administered to 10 patients in remission of acute myeloid leukaemia and three with multiple myeloma 1-4 weeks after treatment with ablative chemotherapy or chemotherapy and autologous bone marrow transplantation. The aim was to assess the capacity of these patients to tolerate IL2 after intensive therapy and to determine whether regenerating lymphocytes were capable of responding to IL2 with the generation of anti-leukaemic effector cells. Toxicity was severe in two patients treated with escalating doses of IL2 and 19 subsequent infusions administered to 11 patients on a fixed dose schedule for periods of 3-5 days were well tolerated. Major toxicity was confined to hypotension (two courses) which responded rapidly to treatment cessation. No patients required intensive care unit support. IL2 infusions produced no significant adverse effects on marrow regeneration; while there were transient falls in platelet counts there were no episodes of clinical bleeding and neutrophil counts increased from a mean of 1.1 pre-infusion to 2.5 x 10(9)l-1 during the infusion (P = 0.004). A significant biochemical abnormality was hypokalaemia which responded rapidly to correction. Cells with activity against leukaemic progenitor cells appeared in peripheral blood within 48 h of beginning treatment. We conclude that IL2 may be used in minimal residual haematological malignancy, and by producing anti-neoplastic effector cells has the potential, as yet unproven, to prolong disease-free survival of patients entering remission.

Although administration of rIL-2 post-T depleted allogeneic bone marrow transplantation (TD-BMT) offers the prospect of augmenting immune reconstitution and thereby reducing the risks of infection and relapse, it has been unclear what direct or indirect effects this agent would have on the regenerating myeloid system. We find that addition of 200 IU or rIL-2 to patient lymphocytes obtained within 6 wk of TD-BMT results in a substantial (2 to 3 log) increase in INF-gamma secretion and the production of TNF. Cytokines present in supernatants obtained from IL-2-stimulated patient lymphocytes have two contrasting effects on myeloid cells from normal donors and from marrow recipients. They prime granulocytes for enhanced oxidative metabolism as measured by ability to generate chemiluminescence in response to FMLP, whereas IL-2 added directly to neutrophils has no effect. However, these IL-2-induced cytokines also act to inhibit myeloid progenitor growth and reduce granulocyte macrophage (GM) colony formation by a mean of 53%. Preincubation of supernatants with anti-IFN-gamma antibody partially abrogates both enhancement of granulocyte chemiluminescence and suppression of marrow CFU-GM. Addition of IL-2 directly to recipient marrow also produces inhibition, leading to a 25% reduction of GM-colony growth. This effect is not due to direct interaction between myeloid progenitor cells and IL-2, because it is completely abrogated by removal of CD8 and Leu-7+ lymphocytes from the marrow. Although the suppressive effects on marrow growth in vitro are of particular concern after BMT, the potential of IL-2 to promote granulocyte function, immune reconstitution, and anti-leukemic activity after TD-BMT justify further consideration of IL-2 therapy in this setting.

Activated killer cells, unrestricted by major histocompatibility (MHC) antigens circulate in the peripheral blood of patients who have undergone autologous and allogeneic bone marrow transplant (BMT) and may contribute to the reduced risk of leukemic relapse observed after these procedures. Interleukin-2 (IL-2) in vitro augments this cytotoxicity and used therapeutically might thereby promote the eradication of minimal residual disease. In order to assess whether these effects on cytotoxicity can be reproduced in vivo, we studied changes in number, phenotype, and MHC unrestricted cytotoxicity of peripheral blood mononuclear cells obtained from patients with hematologic malignancy receiving IL-2 infusions. Patients with acute myeloid leukemia and multiple myeloma were treated after cytotoxic chemotherapy or autologous BMT. IL-2 infusions produced an initial lymphopenia, followed by a progressive recovery in mononuclear cell numbers and a rebound lymphocytosis after the termination of treatment. This affected all lymphocyte subsets; in particular CD25 (IL-2 receptor) positive cell numbers rose sevenfold. Cells with the ability to kill a natural killer (NK)-resistant, lymphokine activated killer cell (LAK)-sensitive target appeared in the circulation during 16 of 19 infusions and mean LAK activity rose from 5.9% to 15.5% during infusion (E:T ratio, 50:1; P less than .001). During IL-2 infusion, cells present in the peripheral blood inhibited the growth of myeloid leukemia blasts in agar after overnight co-culture. Depletion experiments showed that LAK activity was mediated by cells of both CD3- CD16+ (NK derived) and CD3+ CD16- (T derived) subsets. LAK precursor activity in peripheral blood also significantly increased during IL-2 infusion. Increases in major histocompatibility complex (MHC) unrestricted cytotoxicity can be produced by IL-2 infusions in vivo and may result in improved relapse-free survival following chemotherapy or BMT.

Culture of lymphocytes from allograft recipients or from normal donors with Interleukin 2 (IL2) induces high levels of gamma-interferon (gamma IFN) and tumour necrosis factor (TNF) secretion. We now show that production of these two cytokines can also be increased after autologous bone marrow transplantation (BMT) and induced following chemotherapy for haematological malignancy. These effects occur even though the regenerating IL2 responsive lymphocytes in this context have previously been extensively exposed to cytotoxic agents. IL2 induced secretion of gamma-IFN and TNF is higher in autograft recipients than in patients treated by chemotherapy alone. This effect may be related to differences in the phenotypic profile between recovering lymphocytes in the two groups and to an increased degree of prior in vivo lymphocyte activation in the autologous bone marrow transplant recipients. In both groups of patients, IL2 acts on CD3+ T cells and on CD16+ NK cells so that depletion of either subset incompletely abrogates IL2 dependent gamma-IFN secretion. As both TNF and gamma-IFN possess anti-leukaemic and anti-infective activity, enhancement of their secretion by IL2 infusion after autologous bone marrow transplant and induction after chemotherapy may be of therapeutic benefit.

Rodent monoclonal antibodies (MAb) directed against cells of the immune system may be used in vivo for applications including conditioning prior to marrow transplantation and treatment of lymphoid malignancies. Although some MAb may lyse targets by complement fixation, MAb dependent cell mediated cytotoxicity (MAb-DCC) appears to be an important additional effector mechanism. We have investigated the cellular basis of the phenomenon and the response of the effector cells to recombinant cytokines in an attempt to maximize the efficacy of MAb-DCC and thereby increase the therapeutic potency of MAbs. Blood mononuclear cells (PBM) coated with CAMPATH 1G (pan lymphocyte reactive rat IgG2b) MAb were used as targets (T) and autologous lymphocytes or granulocytes were used as effector (E) cells. We studied function in normal donors as well as patients 1 week before and 3-6 weeks after bone marrow transplantation (BMT). In the absence of CAMPATH 1G, specific 51Cr release from autologous PBM was less than 1% in all groups, even after pre-incubation of the effector cells with granulocyte-macrophage, colony stimulating factor (GM-CSF) or interleukin-2 (IL2). In the presence of 5 micrograms/ml of MAb, and at an E:T ratio of 50:1, lymphocytes from normal donors induced a low level (6.5%) of 51Cr release from auto PBM rising to 9% after pre-incubation of effector cells with IL2 (P = 0.02). Granulocytes had greater activity inducing 10% 51CR release (range 2-23%) which rose to 21.6% with GM-CSF (range 12-48%) (P less than 0.001). Pre-BMT, killing by lymphocytes and granulocytes was not significantly different from normal, and responded to IL2 and GM-CSF. In contrast, granulocyte killing after BMT was significantly impaired (51Cr release 3%) and showed no rise with GM-CSF. Killing by lymphocytes, however, remained normal, as did their IL2 response. Loss of granulocyte mediated MAb-DCC coincided with significant post-BMT impairment of oxidative metabolism; expression of Fc receptors II and III, however, was normal. Optimum therapeutic effect of MAb-DCC is likely to be achieved when MAb are given together with appropriate cytokines, the choice of which will depend upon the clinical circumstances.

Interleukin-2 (IL-2) therapy may improve immune reconstitution and reduce the risk of leukemic relapse in the setting of minimal residual disease by augmenting cytotoxic effector mechanisms directed at residual malignant cells. In addition, IL-2 in vitro promotes the release of cytokines including gamma-interferon (gamma-IFN) and tumor necrosis factor (TNF), which also possess antileukemic activity and can enhance granulocyte function. To determine if IL-2 infusion induces release of gamma-IFN and TNF in vivo in sufficient quantity to mediate these effects, we have measured serum levels of these cytokines and secretion by lymphocytes obtained from patients receiving this cytokine in a phase 1 trial. Serum gamma-IFN was undetectable pre-IL-2 and increased to 1.5 to 17 U/mL during IL-2 infusion (P less than .05). Culture of patient lymphocytes for 48 hours produced 1.2 U gamma-IFN/2 x 10(6) cells/mL pre-IL-2 rising to 50 U/2 x 10(6) cells/mL when the lymphocytes were obtained during therapy (P less than .05). Lymphocyte subset analysis showed that both CD3+ and CD16+ cells secreted gamma-IFN in response to IL-2. TNF secretion by lymphocytes also rose during IL-2 infusion from a mean of 5 U/mL to 14.4 U/mL (P less than .01) although no rise was seen in serum levels. The material secreted by IL-2-stimulated lymphocytes is bioactive as addition of supernatants from lymphocytes obtained during IL-2 therapy to cultures of myeloid blasts significantly inhibited clonogenic growth. IL-2-induced secretion of these cytokines mediated this inhibition as it could be partially blocked by either anti-gamma-IFN or anti-TNF antibodies. Preincubation of granulocytes with the same supernatants produced enhanced oxidative metabolism, measured by chemiluminescence in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP). This effect also could be partially abrogated by anti-gamma-IFN and anti-TNF antibodies. Therefore, secondary cytokine secretion may boost granulocyte function and contribute to the antileukemic effects of IL-2 infusion in patients following bone marrow transplantation or chemotherapy.