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An association between auto-immune disorders and interferon (IFN) has been reported. High levels of natural IFNalpha are present in the blood of patients with auto-immune disease and correlate with disease activity. In addition, IFNalpha treatment of humans has resulted in multiple reports of associated auto-immune phenomena. We describe a patient who underwent resection of regionally metastatic melanoma, was given adjuvant high-dose IFNalpha2b, and subsequently developed dermatomyositis. To the authors' knowledge this is the first report of dermatomyositis in association with IFNalpha treatment. We review the literature reporting associations between IFNalpha and auto-immune disease and discuss possible mechanisms by which IFNalpha may contribute to the development of auto-immune disease. High dose IFNalpha2b is more commonly prescribed since it was approved as an adjuvant treatment for patients with surgically resected high-risk melanoma. The potential for cases of IFN-associated auto-immune disease is therefore a clinical concern. Standard side effects of high-dose IFN therapy resemble symptoms of auto-immune diseases, which may make prompt diagnosis difficult. Therefore, it is important that auto-immune diseases such as dermatomyositis are recognized as potential side effects of treatment with high-dose IFNalpha. [ABSTRACT FROM AUTHOR]

Particle-mediated gene delivery was used to immunize mice against melanoma. Mice were immunized with a plasmid cDNA coding for the human melanoma-associated antigen, gp100. Murine B16 melanoma, stably transfected with human gp100 expression plasmid, was used as a tumor model. Particle-mediated delivery of gp100 plasmid into the skin of naïve mice resulted in significant protection from a subsequent tumor challenge. Co-delivery of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) expression plasmid together with the gp100 plasmid consistently resulted in a greater level of protection from tumor challenge. The inclusion of the GM-CSF plasmid with the gp100 DNA vaccine allowed a reduction in the gp100 plasmid dose required for antitumor efficacy. Protection from tumor challenge was achieved with as little as 62.5 ng of gp100 DNA per vaccination. Tumor protection induced by the gp100 + GM-CSF gene combination was T cell mediated, because it was abrogated in vaccinated mice treated with anti-CD4 and anti-CD8 monoclonal antibodies. In addition, administration of the gp100 + GM-CSF DNA vaccine to mice bearing established 7-day tumors resulted in significant suppression of tumor growth. These results indicate that inclusion of GM-CSF DNA augments the efficacy of particle-mediated vaccination with gp100 DNA, and this form of combined gp100 + GM-CSF DNA vaccine warrants clinical evaluation in melanoma patients.

Background: This study was performed to determine the incidence of central venous device-related blood stream infection and thrombosis in patients treated with moderate dose continuous-infusion interleukin-2 (IL-2)., Methods: The records of 160 consecutive patients treated at the University of Wisconsin Hospital and Clinics, between June 1990 and June 1997, with moderate dose continuous-infusion IL-2 (IL-2 [1.5-3.0 x 10(6) U/m(2)/day] Hoffman-LaRoche, Nutley, NJ or IL-2 [4.5 x 10(6) U/m(2)/day] Chiron Corporation, Berkley, CA) were reviewed retrospectively. The majority of patients had metastatic melanoma (78 patients) or renal cell carcinoma (70 patients). All of the patients had a surgically implanted central venous device placed before starting IL-2 therapy; 89% of these were cuffed Hickman catheters. Eighty-four patients received 1 mg of warfarin per day as prophylaxis against device-related thrombosis; none received periinsertion prophylactic antibiotics., Results: Twenty-one patients (13%) developed central venous device-related bloodstream infection (DRBSI) during the study period, a rate of 2 DRBSI per 1000 device-days. DRBSIs were associated with the type of immunotherapy given with IL-2 (P = 0.01) and with thrombosis (odds ratio, 4.1; 95% confidence interval, 1.5-11.4; P = 0.008) but not with patient gender, type of cancer, duration of the central device, or site of device placement. Twenty-six patients (16%) developed central venous device-related thrombosis (DRT) during immunotherapy. Low dose warfarin did not appear to prevent thrombosis. Device-related thrombosis was associated with DRBSI but not with patient gender, type of cancer, type of device, duration or location of device, or concomitant immunotherapy., Conclusions: Central venous DRBSI and DRT are significant complications that can occur during moderate dose continuous-infusion IL-2 therapy. The risk of DRBSI appears lower than the risk reported with high dose IL-2 therapy by previous investigators. The risk of DRT appears to be higher than the risk reported for patients with similar devices but not given IL-2. Low dose warfarin did not prevent DRT when started after device placement., (Copyright 2001 American Cancer Society.)

IL-12 gene therapy results in tumor regression in some, but not all, murine models. We hypothesized that expression of B7.1 on the tumor cell surface was necessary for IL-12-mediated tumor regression. In addition, we hypothesized that all cells must express B7.1 for this to be effective. To evaluate this hypothesis, tumor nodules were established in mice with either wild-type B16 melanoma or with B16 melanoma modified to express B7.1. IL-12 cDNA was transferred to the tumor by particle-mediated gene transfer. All tumors modified to express B7.1 regressed completely after IL-12 cDNA treatment. When the percent of B7.1-transfected B16 cells was decreased to 50%, no animals survived after treatment. Animals rendered tumor-free were then challenged with wild-type B16. Fifty percent of mice was protected from this tumor challenge. Expression of CD28 (the stimulatory B7.1 ligand) was significantly increased in both CD8(+) T cells and natural killer cell populations of mice rejecting tumor challenge compared to mice with tumor growth. These results suggest that the costimulatory molecule B7.1 is required for initial tumor sensitivity to IL-12 gene therapy and that protection from subsequent challenge with B7.1 (-) tumor is mediated by CD28(+) immune effector cells.

The aim of this study was to compare the antigenicity of human melanoma cells molecularly modified by particle-mediated gene transfer to have transient or stable expression of the B7-1 co-stimulatory molecule (CD80). The unmodified melanoma cells (mel5, m21) had no constitutive expression of B7-1, but 22%-28% of cells had transient B7-1 expression 24 h following transfection with cDNA for B7-1 (mel5-B7, m21-B7). In addition, 85%-90% of cells had stable B7-1 expression following transfection with cDNA for B7-1 and in vitro culture under selection conditions (mel5-B7neo, m21-B7neo). Allogeneic HLA-unmatched normal donor peripheral blood mononuclear cells (PBMC) secreted greater amounts of granulocyte/macrophage-colony-stimulating factor (GM-CSF) when incubated for 3 days with m21-B7neo than did PBMC incubated with m21-B7, which, in turn, secreted greater amount of GM-CSF than PBMC incubated with m21. Similarly, cell-mediated cytotoxicity against unmodified melanoma cells by PBMC co-cultured for 5 days with the modified or unmodified melanoma cells was proportional to the level of B7-1 expression on the stimulating cells. This cytolytic activity had both an HLA-class-I-restricted and an HLA-class-I-unrestricted component. Following 5 days of co-culture, PBMC expression of CD28, the ligand for B7-1, was down-regulated in proportion to the level of B7-1 expression on the stimulating melanoma cells. Thus, particle-mediated gene delivery of cDNA for B7-1 into human melanoma cells increased expression of functional B7-1 and enhanced the antigenicity of the gene-modified cells in proportion to their level of B7-1 expression.

We conducted a Phase IB trial of antidisialoganglioside chimeric 14. 18 (ch14.18) antibody and interleukin 2 (IL-2) to determine the maximal tolerated dose (MTD), immunological effects, antitumor effects, and toxicity of this treatment combination. Twenty-four melanoma patients received immunotherapy with ch14.18 antibody and a continuous infusion of Roche IL-2 (1.5 x 10(6) units/m2/day) given 4 days/week for 3 weeks. The ch14.18 antibody (dose level, 2-10 mg/m2/day) was scheduled to be given for 5 days, before, during, or following initial systemic IL-2 treatment. The ch14.18 MTD was 7.5 mg/m2/day, and 15 patients were treated with the ch14.18 MTD. Immunological effects included the induction of lymphokine-activated killer activity and antibody-dependent cellular cytotoxicity by peripheral blood mononuclear cells. In addition, serum samples obtained following ch14.18 infusions were able to facilitate in vitro antibody-dependent cellular cytotoxicity. Antitumor activity included one complete response, one partial response, eight patients with stable disease, and one patient with >50% decrease of hepatic metastases in the face of recurrence of a s.c. lesion. Dose-limiting toxicities were a severe allergic reaction and weakness, pericardial effusion, and decreased performance status. Most patients treated at the MTD had abdominal, chest, or extremity pain requiring i.v. morphine. One patient had an objective peripheral neuropathy. This IL-2 and ch14.18 treatment combination induces immune activation in all patients and antitumor activity in some melanoma patients. We are attempting to enhance this treatment approach by addition of the anti-GD3 R24 antibody to this IL-2 and ch14.18 regimen.

The induction of human antimouse antibodies (HAMA) and human anti-idiotypic (anti-Id) responses in cancer patients receiving therapeutic monoclonal antibody (mAb) may limit the effectiveness of the administered mAb. This report evaluates the influence of systemic interleukin-2 (IL-2) on the anti-Id response to anti-disialoganglioside (anti-GD2) antibody given as treatment for patients with melanoma. Twenty-eight patients with melanoma received combined immunotherapy with anti-GD2 antibody and IL-2 at 1.5 x 10(6) U/m2/day given 4 days/week. The anti-GD2 antibody [murine 14.G2a mAb; dose levels of 2-5 mg/m2/day (4 patients); or human-mouse chimeric 14.18 (ch14.18) antibody; dose levels of 2-10 mg/m2/day (24 patients)] was scheduled to be given for 5 days either before, during, or after initial systemic IL-2 treatment. All four patients who received murine 14.G2a developed HAMA anti-isotype antibodies (660-1,000 ng/ml) as well as measurable anti-Id antibodies. All three patients who received initial treatment with ch14.18 alone developed a strong anti-Id antibody response after IL-2 was started 1 week later. The serum level of anti-Id antibody decreased during subsequent ch14.18 infusions, suggesting that the anti-Id antibody may be binding the administered ch14.18. In contrast, measurable anti-Id antibody was detected in only 3 of 14 patients who received IL-2 before, during, and after initial ch14.18 administration. Two of four patients receiving systemic IL-2 before and during initial ch14.18 infusions, and two of three patients receiving systemic IL-2 concurrent with initial ch14.18 infusions developed anti-Id antibodies. These data suggest that the anti-Id response to chimeric anti-GD2 antibody is influenced by the timing of systemic IL-2 in relation to antibody administration and can be suppressed by systemic treatment with IL-2 given before, during, and after the antibody administration.

The aim of this study was to determine if human melanoma cells could be molecularly modified by particle-mediated gene transfer with a "gene gun", using genes for interferon-gamma (IFN-gamma), the B7-1 costimulatory molecule (CD80), and human leukocyte antigen (HLA)-A2, to augment expression of both HLA molecules and B7-1. Established and early passage melanoma cells transfected with human IFN-gamma complementary DNA (cDNA) produced IFN-gamma (50-5,000 pg/mL). The biological effect of this IFN-gamma transgene included an upregulation, or de novo appearance, of HLA expression. These melanoma cells had no detectable baseline surface expression of the B7-1 costimulatory molecule, but 8% to 31% of these cells became B7-1 positive with no selection procedure after gene transfer with human B7-1 cDNA. After combination gene transfer with cDNAs for both IFN-gamma and B7-1, 9% to 33% of these cells expressed both HLA-DR and B7-1. In combination gene transfer experiments with cDNAs for both HLA-A2 and B7-1, dual expression of HLA-A2 and B7-1 was achieved in 10% to 17% of the melanoma cells. Thus, the molecular modification of human melanoma cells to increase expression of both HLA and B7-1 can be achieved by particle-mediated gene delivery and presents a promising strategy to stimulate antimelanoma T-cell immunity. Key words: Melanoma; T cells; B7-1 costimulatory molecule (CD80); major histocompatibility complex.

Renal cell carcinoma (RCC) is relatively resistant to chemotherapy and radiotherapy, whereas treatment with biologics has achieved limited success. Although monoclonal antibodies able to recognize human RCC have been identified, most induce little complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity (ADCC), and thus are of limited potential as therapeutic modalities in their natural conformation. We evaluated a human/ mouse chimeric derivative of the previously described G250 murine monoclonal antibody (mAb), reactive with RCC, to identify a reagent for potential immunotherapy. This chimeric antibody (ch-G250) is composed of the murine variable region from the G250 mAb, which recognizes a tumor-associated antigen expressed on 95% of primary and 86% of metastatic renal cell carcinomas. The constant region of the ch-G250 is comprised of the human IgG1 isotype domains. This chimeric antibody does not bind to normal renal tissue or other normal human tissues, with the exception of gastric mucosal cells and large bile-duct epithelium. Clinical radiolocalization studies have demonstrated the relative tumor-targeting potential of this radiolabeled antibody. This ch-G250 antibody facilitated potent ADCC against several RCC lines when using in vitro and in vivo interleukin-2 (IL-2)-activated peripheral blood mononuclear cells obtained from healthy control donors and patients with cancer, respectively. This lymphocyte-mediated ADCC was specific for RCC cells recognized by the ch-G250 antibody. Using flow cytometry, we found that the level of ADCC was directly related to the degree of binding of ch-G250 to the renal cell target. These in vitro data suggest that this antibody may improve efficacy of IL-2 therapy by targeting cytokine-activated effector cells directly to the tumor and facilitating in vivo ADCC. Clinical studies combining this chimeric antibody with IL-2 treatment will be needed to test the antitumor effects of this ADCC effect in vivo.

Interleukin 2 (IL-2) and granulocytes-macrophage colony-stimulating factor (GM-CSF) are activators of the lymphocyte and granulocyte/macrophage series, respectively. We conducted a phase IB trial to identify the maximally tolerated dose and to assess immunological effects of the combination. Thirty-four patients with incurable cancers received 2.5, 5, or 10 microgram/kg GM-CSF s.c. either before or concurrently with 1.5 or 3.0 million units/m2/day IL-2. The most common laboratory and clinical side effects included an elevation of the total WBC or eosinophil count due to GM-CSF, and constitutional symptoms due to IL-2. Grade 3 or 4 toxicities included hypotension, thrombocytopenia, elevations in aspartate aminotransferase or bilirubin, renal toxicity, gastrointestinal hemorrhage, arrhythmia, and constitutional symptoms. Two patients receiving 5.0 microgram/kg GM-CSF plus concurrent 3.0 million units IL-2 experienced dose-limiting grade 3 or 4 neurological toxicity, which reversed almost completely. An increase in the serum-soluble IL-2 alpha chain receptor was observed with administration of GM-CSF, IL-2, or the combination. IL-2 therapy enhanced lymphokine-activated killer activity, antibody-dependent cellular cytotoxicity, and lymphocyte activation, with increased CD16 and CD56 expression. GM-CSF increased expression of human leukocyte antigen DR on peripheral blood monocytes and decreased surface expression of CD16 on circulating monocytes and polymorphonuclear cells. Lymphokine-activated killer activity and CD16 expression on monocytes and lymphocytes and CD56 expression on lymphocytes were significantly lower in patients receiving GM-CSF simultaneously with IL-2 than in patients receiving the sequential treatment. Antitumor activity was observed in the lungs of four of eight renal cell carcinoma patients with pulmonary metastases treated with concurrent GM-CSF and IL-2. Although no or minimal shrinkage was observed in the patients' large primary tumors, these results warrant further study. The recommended initial Phase II dose and schedule is 1.25 microgram/kg/day GM-CSF, given concurrently with 1.5 million Roche units/m2/day (4.5 x 10(6) international units/m2/day) IL-2, with subsequent escalation of GM-CSF to 2.5 microgram/kg/day after careful observation for toxicities.

Background: Splenomegaly in patients with cancer raises the suspicion of tumor involvement. Splenic enlargement in the absence of splenic metastases, however, has been reported in patients treated with interleukin-2 (IL-2) immunotherapy. This study characterizes the change in spleen size that occurred in 42 patients treated with IL-2 between 1989 and 1993 for nonhematologic malignancies., Methods: Computed tomography (CT) scans before and during immunotherapy were available for review on all 42 patients and after immunotherapy on 16 of these patients. The splenic index was measured for each CT by a single reader blinded to the time course of IL-2 therapy., Results: Mean splenic index increased 64.1% from 646 cm3 (standard deviation [SD], 387) pre-IL-2 to 1059 cm3 (SD, 534) during therapy with IL-2 (P < 0.0001). The splenic index remained elevated at 1112 cm3 (SD, 633) after completion of IL-2 therapy., Conclusions: Splenomegaly, temporally associated with IL-2 therapy for nonhematologic malignancies, is likely to represent a sequela of therapy and not tumor progression.

Immunocompetent cells in bone marrow allografts have been associated with a graft-versus-leukemia (GVL) effect. To further characterize effector mechanisms that may be involved in this GVL phenomenon, we have previously established an in vitro model to identify allogeneic T-cell clones that selectively mediate cytotoxicity against a patient's leukemic cells, but not against nonleukemic lymphocytes from the same patient. We have modified this in vitro model to test whether the Ph1 chromosome and the P210 fusion protein it controls have a detectable role in leukemia-specific recognition by allogeneic T-cell clones. In this report, T-cell lines reactive with allogeneic Ph1 chromosome-bearing (Ph1+) chronic myeloid leukemia (CML) cell lines were derived and selected to be minimally reactive with Ph1 negative (Ph1-) lymphoid lines from the same patient. However, after prolonged culture, these same T-cell lines also mediated significant destruction of the Ph1- target cells from the same patients. These T-cell lines specifically recognized cells from the allogeneic CML patient to which they were sensitized, and were not contaminated by an outgrowth of natural killer cells. Furthermore, subclones could be derived from these T-cell lines, and some of these subclones again showed selective killing of the allogeneic Ph1+ leukemia cell lines, and not of the Ph1- cell line from the same patient. Analyses of T-cell receptor (TCR) genes showed the alloreactive T-cell lines and the Ph1+ selective subclones derived from them to be of the same clonal origin. This suggests that the same T cells reacting with antigens expressed on the nonleukemic Ph1- targets can at times selectively and preferentially kill the allogeneic Ph1+ cells. As the same TCR that recognizes Ph1+ cells also can recognize the Ph1- targets, it appears that the Ph1+ chromosome does not play a detectable role in recognition by these allogeneic T-cell clones. This in vitro observation may provide a model for evaluating the relationship between GVL and graft-versus-host disease effects.

Recent technical advances have enabled the generation of clinical reagents for immunotherapy. Currently, treatment protocols combining both interleukin-2 (IL-2) and tumor-specific monoclonal antibody are underway at the University of Wisconsin Comprehensive Cancer Center and elsewhere. These approaches are based on the hypothesis that IL-2-activated lymphocytes will use tumor-reactive antibody to more selectively and effectively destroy tumor in vivo. Just as IL-2 can activate lymphocytes to destroy antibody-coated tumor cells, other agents can activate neutrophils and monocytes to destroy antibody-treated tumor cells. We are investigating, in laboratory and clinic, approaches aimed at eventually using combinations of distinct antibody-based tumor recognition mechanisms in patients whose monocytes, neutrophils, and lymphocytes have been simultaneously activated with multiple biologic agents.

The culture conditions of the immortalized human breast cell line, 184B5, can be manipulated to evaluate conditions that influence target lysis by activated immune effector cells. Exponentially growing 184B5 cells (EXP) are more susceptible than growth-factor-deprived (removal of epidermal growth factor and bovine pituitary extract) 184B5 cells (GFD) to lysis by lymphokine-activated killer (LAK) cells, activated natural killer (NK) clones, and activated gamma/delta receptor expressing T-cell clones. LAK-cell lysis of contact-inhibited 184B5 cells is similar to lysis of GFD, while 184B5 cells with severe nutrient deprivation are more easily lysed than GFD by LAK cells. In a cold target inhibition assay with LAK effector populations, EXP are better inhibitors than GFD against several targets. Further analysis of the mechanism by which changes of in vitro culture conditions alter target-cell susceptibility to immune-mediated lysis may assist therapeutic strategies that involve combinations of standard therapies with biologic approaches.

Nine patients with metastatic renal cell carcinoma were entered into a pilot protocol including a 4-week regimen utilizing human recombinant interleukin-2 (IL-2) and in vitro lymphokine-activated killer (LAK) cells. The regimen included 2 weeks (4 days of treatment and 3 days of rest/week) of continuous-infusion (c.i.) IL-2 at 3 x 10(6) U/m2/day, followed by two leukaphereses. LAK cells were cultured in vitro for 48 to 72 h and administered as a single infusion, followed by 9 days of bolus i.v. injections of 10(6) U IL-2/m2/dose, given every 8 hours (t.i.d.). The average (+/- SD) number of LAK cells infused per patient was 7.2 x 10(10) (+/- 3.5 x 10(10)). One patient showed > 50% shrinkage of tumor (lung + renal bed recurrence). Toxicity was similar to that encountered in other studies using similar IL-2 doses and LAK cells and consisted of fever, hypotension, fluid retention, and reversible renal insufficiency. These results indicate that the 2 weeks of IL-2 c.i. provided conditions enabling the harvest of large quantities of mononuclear cells from the peripheral blood of patients; this could be useful for future trials requiring the use of in vitro activated lymphocytes. Nevertheless, these pilot data suggest that this regimen of prolonged t.i.d. IL-2 administration after the LAK infusion does not seem to generate any improvement in antitumor effects from those obtained using other LAK + IL-2 regimens.

The design of combination hormonal and immunotherapeutic protocols for breast cancer patients may be facilitated by analysis of preclinical in vitro model systems. Estrogen receptor positive (ER+: MCF-7) and negative (ER-: MDA-MB-231) human breast cancer cell lines were utilized to evaluate the effects of tamoxifen (TAM) and estradiol (E2) on modulation of breast cancer target susceptibility to lysis by lymphokine-activated killer (LAK) cells. E2-stimulated ER+ cells were more susceptible to lysis by LAK cells than corresponding TAM-treated or control cells, while treatment of ER- cells with either E2 or TAM alone did not alter from control their susceptibility to this immune-mediated lysis. All ER+ and ER- cells tested remained sensitive after treatment with TAM to lysis by LAK cells. In addition, an adenocarcinoma reactive human-mouse chimeric monoclonal antibody (ING-1) was able to significantly boost in vivo generated LAK cell-mediated lysis of control, E2-treated, and TAM-treated ER+ and ER- cells. These in vitro results provide a preclinical rationale for in vivo testing of TAM, interleukin-2 (IL-2), and breast cancer reactive antibody-dependent cellular cytotoxicity facilitating antibody in patients with refractory or high risk breast cancer.

A total of 199 T cell clones from two melanoma patients were derived from progenitor T cells from recurrent melanoma, regional lymph nodes (either involved or uninvolved with malignancy) and peripheral blood by inoculating single cells directly into the wells of microtiter plates before in vitro expansion. The surface marker phenotype of most clones was CD4+CD8-, although some were CD4-CD8+. Genomic DNA prepared from all clones was analyzed by Southern blot hybridization using T cell receptor (TCR) beta and gamma gene probes, seeking clones with identical TCR gene rearrangement patterns as direct evidence for in vivo progenitor T cell clonal amplification. Probing HindIII-digested DNA with TCR beta and TCR gamma probes revealed several clones with identical TCR gene rearrangement patterns. These clones had subsequent probing of BamHI-digested DNA with TCR beta and TCR gamma probes, which showed all but 2 clones to have distinct rearrangement patterns. These analyses provide clear molecular evidence for in vivo polyclonal CD4+ T cell populations in each of several separate immune compartments in these patients.

Preliminary studies involving small numbers of patients have suggested that interleukin-2 (IL-2) administered by continuous infusion in repetitive weekly cycles using doses of 3 x 10(6) U/M2/day is immunologically active and can induce tumor responses in patients with renal cell carcinoma. This study was designed to examine both the immunological and clinical effects of prolonged infusion IL-2 given by repetitive weekly cycles; first at moderate doses for 4 weeks as an impatient followed by lower doses of IL-2 for up to 5 months. Prolonged IL-2 treatment was investigated because previous studies revealed that patients had a return to their baseline immune status within 4 weeks after completing IL-2 treatment. Twenty-five patients (including 18 with renal cell carcinoma) were treated with one of two regimens utilizing IL-2 as sole therapy. These regimens were designed to induce augmented and prolonged immune activation based upon in vitro and in vivo data. Though patients on both arms of the study demonstrated sustained lymphocytosis, increase in numbers of natural killer cells, and induction of lymphokine-activated killer activity with prolonged IL-2 administration, only 1 out of the 18 patients with renal cell carcinoma demonstrated a sustained partial antitumor response to therapy. Furthermore, several patients demonstrated profound immune activation, without any evidence of tumor regression. The lack of clinical responses in these patients showing marked activation of LAK cytotoxicity suggests that other variables must also influence the likelihood of antitumor effects for patients receiving IL-2 therapy.

Eleven patients receiving weekly cycles of therapy with recombinant interleukin-2 (IL-2) were evaluated with a sensitive limiting dilution analysis to determine lymphokine-activated killer (LAK) cell precursor frequencies in peripheral blood lymphocytes. An increase in LAK precursor frequency above baseline was suggested by day 6 of this protocol and was clearly significant by day 20, indicating an expansion of the circulating precursor pool results from in vivo IL-2 administration. Correlations were not significant between LAK precursor frequency during IL-2 therapy and the total number of circulating lymphocytes, the percentage of CD56+ lymphocytes, IL-2 proliferative responses, or LAK activity of peripheral blood lymphocytes, indicating that the precursor frequency identification based on functional testing of individual cells is not accurately reflected by these analyses of heterogeneous bulk populations. Selective cell depletion analyses revealed that the majority of LAK precursors after in vivo IL-2 therapy were cells with the natural killer phenotype. Analysis of LAK precursors may help define the in vivo IL-2 administration, alone or in combination with other hematopoietic or immunodifferentiative cytokines, necessary to further augment in vivo effector cell numbers and activity for patients with cancer.