Published ahead of print on April 17, 2003, doi:10.1164/rccm.200211-1359OC
© 2003 American Thoracic Society
Randomized Trial of Adjunctive Interleukin-2 in Adults with Pulmonary TuberculosisDivision of Infectious Diseases, Department of Medicine, Case Western Reserve University School of Medicine and University Hospitals of Cleveland, Cleveland, Ohio; University of Arkansas for Medical Sciences, Little Rock, Arkansas; Division of Infectious Diseases, Department of Medicine, University of Medicine and Dentistry of New Jersey, Newark, New Jersey; Department of Medicine, Mulago Hospital and Makerere University; and National TB and Leprosy Control Programme, Kampala, Uganda Correspondence and requests for reprints should be addressed to John L. Johnson, M.D., Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, Room E-202, Tuberculosis Research Unit, 10900 Euclid Avenue, Cleveland, OH 44106–4984. E-mail: jlj{at}po.cwru.edu
Interleukin (IL)-2 has a central role in regulating T cell responses to Mycobacterium tuberculosis. Adjunctive immunotherapy with recombinant human IL-2 was studied in a randomized, placebo-controlled, double-blinded trial in 110 human immunodeficiency virus–seronegative adults in whom smear-positive, drug-susceptible pulmonary tuberculosis was newly diagnosed. Patients were randomly assigned to receive twice-daily injections of 225, 000 IU of IL-2 or placebo for the first 30 days of treatment in addition to standard chemotherapy. Subjects were followed for 1 year. The primary endpoint was the proportion of patients with sputum culture conversion after 1 and 2 months of treatment. After 1 month, the proportion of patients for whom sputum culture converted to negative was 17% for the IL-2 group compared with 30% in the control group (p = 0.14;  2). After 2 months, 77% in the IL-2 group were culture negative compared with 85% of those receiving placebo (p = 0.29, 2). Results were similar when patients with isoniazid monoresistance were included in the analysis. There were no differences in weight gain and no improvement in fever, cough, and chest pain between groups. One patient in each arm relapsed. IL-2 did not enhance bacillary clearance or improvement in symptoms in human immunodeficiency virus–seronegative adults with drug-susceptible tuberculosis.
Key Words: tuberculosis, pulmonary • antitubercular agents • immunotherapy • interleukin-2 Tuberculosis (TB) is a major global health problem. Up to one-third of the world's population is infected with Mycobacterium tuberculosis. The World Health Organization estimates that 8 million new TB cases and 1.9 million deaths due to TB occurred worldwide in 1997 (1). In addition, drug resistance to standard anti-TB drugs has increased in many areas during the past decade (2). New modalities for the prevention and treatment of TB are clearly needed. Recovery from TB depends, in part, on the generation of an effective cell-mediated immune response against the pathogen. Effective T cell function is key in controlling M. tuberculosis infection. Interleukin (IL)-2, a cytokine produced by activated T lymphocytes, has a central role in the activation and expansion of T cells. In murine models of Mycobacterium lepraemurium, Mycobacterium avium and Mycobacterium bovis bacillus Calmette–Guérin infection, IL-2 has been shown to limit mycobacterial replication, possibly by macrophage activation via interferon-mediated pathways or directly by the development of cytotoxic T lymphocytes recognizing mycobacterial antigens (3–5). Patients with TB frequently have deficient IL-2–induced cell proliferation and decreased IL-2 receptor generation (6). These observations form the basis for studies of recombinant IL-2 as adjunctive immunotherapy against mycobacterial diseases in humans. Early clinical trials with IL-2 in patients with leprosy, leishmaniasis, TB and other serious infections due to intracellular pathogens demonstrated that IL-2 immunotherapy may be useful in controlling these infections (7–14). In patients with leprosy, IL-2 administration led to enhanced local cell-mediated immune responses and resulted in more rapid and extensive reduction in bacilli compared with multidrug chemotherapy alone (11, 12). In a pilot study from Bangladesh and South Africa, treatment of patients in whom drug-sensitive and chronic multidrug-resistant (MDR) TB was newly diagnosed with 12.5 µg (225, 000 IU) of intradermal IL-2 twice daily during the first month of TB therapy resulted in rapid sputum conversion (13). A later randomized trial in South Africa comparing daily and pulsed IL-2 with placebo in MDR TB found improved sputum clearance with daily treatment (14). These results suggested a potential role for IL-2 in TB treatment. To further study this issue, we conducted a randomized, double-blinded, placebo-controlled Phase II clinical trial to evaluate the safety and microbiologic and immunologic effects of IL-2 in human immunodeficiency virus–seronegative adults with initial episodes of smear-positive, drug-susceptible pulmonary TB. We hypothesized that adjunctive treatment with IL-2 would enhance cell-mediated immune responses in TB and increase the rate of killing of tubercle bacilli and that these effects may be most evident during early treatment in patients with drug-susceptible TB. Some of the results of this trial have been previously reported in the form of an abstract (15).
Patients Ambulatory patients, aged 18 to 50 years, with suspected pulmonary TB were referred from the main outpatient clinic of the National TB Treatment Centre, Mulago Hospital, Kampala, Uganda for possible study participation. Human immunodeficiency virus–seronegative patients in whom initial episodes of smear-positive, culture-confirmed TB was newly diagnosed and who had moderately advanced or far-advanced TB on chest X-ray (16), a Karnofsky performance scale score greater than 50% (17), and were not pregnant or lactating were eligible for the study. Persons previously treated for TB, patients with asthma, untreated thyroid disease, or other serious medical conditions, those with a hemoglobin less than 80 g/L, total white blood cell count less than 3,000/mm3, serum aspartate aminotransferase greater than 100 IU/L or serum creatinine more than 177 µM/L or limited respiratory reserve, and persons on chronic corticosteroid or immunosuppressive drugs were excluded. Patients found to have initial drug resistance to isoniazid (INH), rifampin, ethambutol or pyrazinamide were excluded from the study when susceptibility testing results became available and their TB treatment was adjusted accordingly. The study was approved by the institutional review boards at University Hospitals of Cleveland and Case Western Reserve University and the Ugandan National AIDS Research Subcommittee. All participants gave informed consent.
Treatment Allocation and Masking A computer-generated randomization sequence with a block size of 10 was used to assign subjects to study treatment. Clinical and laboratory staff were masked to treatment assignment. Separate clinical assessors were used to assess local and systemic adverse experiences. Treatment assignments were not revealed to any investigator or subject during the trial. Additional details are available in the online supplement.
Baseline Measurements
Bacteriology.
Immunologic measurements. Sera were stored before beginning treatment and after 2, 4, and 6 weeks of anti-TB treatment for measurement of anti–IL-2 antibodies. Detailed procedures for the microbiologic and immunologic assays are included in the online supplement.
Anti-TB Chemotherapy
Follow-up Measurements Sputum was collected for acid-fast bacilli smear and culture after 2 and 4 days of treatment, then weekly from weeks 1 through 4, after 6 weeks, and then monthly during the remainder of treatment as long as the subject was able to produce sputum. Immunologic measurements were repeated after 2 weeks and 6 weeks of anti-TB treatment. Subjects were followed for 1 year after the onset of anti-TB treatment.
Statistical Analysis
The total sample size (110) was calculated to have 80% power (
All statistical analyses were performed using SAS software (SAS, version 6.12, SAS Institute, Cary, NC). Significant univariate differences between the two study arms were determined using Studies of the British Medical Research Council with rifampin-containing short-course chemotherapy regimens suggested that INH monoresistance or resistance to INH plus streptomycin had little effect on sputum culture conversion after 2 months of treatment and relapse (26). We therefore also analyzed our data combining those subjects with INH monoresistance with those with fully drug-susceptible TB.
Study Population Five hundred fifty-four adult volunteers with suspected initial episodes of pulmonary TB were evaluated for study participation. One hundred and ten patients were enrolled and randomized to study treatment (Figure 1) . Fifty-five patients received IL-2 immunotherapy and 55 patients received placebo.
Table 1 shows the characteristics of all subjects at the time of randomization. Patients randomized to IL-2 were less likely to have a bacillus Calmette–Guérin scar and report chest pain or night sweats. These variables were explored for potential confounding and interaction; no strong evidence for either was found. Twenty-five percent of all subjects had moderately severe TB on chest X-ray and 75% had far-advanced disease. Ninety-six percent had cavitary disease. Ninety-two percent of all subjects had grade 3+ or 4+ sputum acid-fast bacilli smears at entry.
One hundred nine subjects received all 60 injections of study drug treatment. Injections were stopped early (after 32 injections) in one subject in the placebo arm who was found to have diabetes mellitus and was transferred from the TB ward to the medical ward for diabetic control. Fifteen enrolled subjects were later terminated from the study or declared ineligible after review of initial drug susceptibility testing results—12 due to INH monoresistance (seven subjects randomized to the IL-2 arm and five in the placebo arm), two subjects in the placebo arm with INH and rifampin resistance, and one subject in the placebo arm who was found to be human immunodeficiency virus–seropositive after enrollment. As specified in the study protocol, these subjects were followed but were excluded from the primary clinical, radiographic, microbiologic, and immunologic analyses. None of these reasons for exclusion differed significantly between treatment groups. All enrolled subjects were included in the safety analysis. The number of patients completing each phase of the study is shown in Figure 1.
Compliance with Standard Short-Course Chemotherapy
Microbiologic Outcomes
Quantitative sputum colony counts also were performed during anti-TB treatment. At several time intervals after initiation of treatment, the sputum bacillary load was lower in subjects receiving placebo compared with IL-2 (p = 0.04 after 4 days, p = 0.02 after 3 weeks, and p = 0.03 after 4 weeks; t test, Figure 3) .
Clinical and Radiographic Outcomes No treatment failures or deaths occurred. One bacteriologically confirmed relapse occurred in each treatment group. For both subjects who relapsed, the sputum M. tuberculosis isolate obtained at the time of relapse was identical to the patient's baseline isolate when compared by IS6110 DNA genotyping (27). One subject who relapsed had initial resistance to INH. Neither subject who relapsed had acquired drug resistance. There were no significant differences in the rate of weight gain, rate of defervescence, improvement in Karnofsky performance scale score, and improvement in self-reported cough and chest pain during TB treatment between groups (data not shown). Radiographic improvement in the extent of disease by one or more severity grades (16) comparing baseline and follow-up chest X-rays after 1, 2, 6, and 12 months of TB treatment did not differ between treatment arms (data not shown). These results were unchanged when patients with INH monoresistance were included in the analysis. Data describing radiographic changes during treatment are included in Table E3 in the online supplement.
Immunologic Changes during Anti-TB Treatment Flow cytometric analysis of peripheral blood cells stained with a combination of monoclonal antibodies to CD3, CD4 or CD8, and CD25 (to assess activation of T cell subsets) or CD3, CD16, and CD56 (to determine frequencies of NK cells) were performed at baseline and after 2 and 6 weeks of anti-TB treatment. The 6-week time point was chosen to assess ongoing immune activation 2 weeks after the end of study drug treatment. The median percentage of CD4+/CD25+ T lymphocytes was greater after 2 and 6 weeks of anti-TB treatment in subjects receiving IL-2 than placebo (p = 0.05 and p = 0.08, Mann–Whitney U test, Figure 4) . By contrast, the percentage of CD8+/CD25+ cells was not increased with IL-2 treatment (data not shown). Systemic activity of IL-2 at Week 2 of study was further corroborated by increased levels of sIL-2R (by ELISA) in serum from patients in the IL-2 compared with the placebo arm (p = 0.004, Mann–Whitney U test, Figure 4). In contrast to previously published results (14), the percentage of CD3-/CD16+/CD56+ NK cells was not increased among peripheral blood mononuclear cells from subjects in the IL-2 arm compared with the placebo arm (data not shown).
The results for the above immunologic measurements were comparable when patients with initial INH resistance were included in the analysis.
Safety
In this randomized placebo-controlled clinical trial, we found that adjunctive immunotherapy with recombinant human IL-2 did not enhance sputum bacillary clearance or improvement in important clinical symptoms in human immunodeficiency virus–seronegative adults with drug-susceptible pulmonary TB. Intradermal therapy with IL-2 was generally safe and well tolerated. The study population included primarily patients with advanced cavitary TB and high sputum bacillary loads where an effect of adjunctive immunotherapy would most likely be evident. Two earlier trials of adjunctive IL-2 in pulmonary TB have been reported (13, 14). In Bangladesh and South Africa, 20 patients who were partially treated, had MDR TB, or were newly diagnosed for TB received 30 days of twice-daily intradermal injections of 225,000 IU (12.5 µg) of IL-2 in addition to chemotherapy (13). All patients in whom TB was newly diagnosed and five of seven patients with MDR TB converted their sputum smears to negative. An increase in PPD skin test size and enhanced T cell responses were seen in patients with drug-susceptible TB. A clinical trial in 35 patients with MDR TB from South Africa compared daily or pulsed IL-2 therapy with placebo (14). Patients received susceptibility-directed chemotherapy and were randomized to receive daily (225,000 IU IL-2 intradermally twice daily), pulsed (three cycles of 450,000 IU IL-2 twice daily for 5 days, followed by 9 days off IL-2), or placebo during the first 30 days of TB treatment. Among smear-positive patients, five of eight patients receiving daily IL-2 had reduced or negative sputum smears compared with two of seven subjects receiving pulsed IL-2 and three of nine subjects in the placebo group. The numbers of IL-2 receptor–positive T cells and of NK cells were increased in patients receiving daily IL-2 but not in the pulsed IL-2 or placebo arms. Chest X-ray improvement after 6 weeks of TB treatment also was more frequent in patients receiving daily IL-2. No significant side effects of IL-2 treatment were observed. We were unable to confirm these earlier reports of a positive effect of adjunctive immunotherapy with IL-2 on clinical, bacteriologic, and radiographic responses in patients who were partially treated, had MDR TB, and were newly diagnosed for TB. The IL-2 treatment regimen used in our study, 225,000 IU intradermally twice daily for the first 30 days of TB treatment, was identical to that used in the randomized clinical trial performed in patients with MDR TB in South Africa by Johnson and colleagues, where IL-2 enhanced sputum smear clearance and radiographic improvement (14). In that study, IL-2 treatment was associated with an increase in peripheral blood of IL-2 receptor–bearing T cells and NK cells. Like Johnson and colleagues, we were able to demonstrate a modest systemic effect of IL-2 as evidenced by an increase in the percentage of CD25-positive CD4 T cells and an increase of levels of sIL-2R and IL-2 in serum in our trial; however, the effect was transient. Thus, the lack of a clinical response to IL-2 therapy may be the result of the transient nature of its effect on immune parameters. However, recent evidence from the literature indicates that a successful anti–M. tuberculosis immune response involves both CD4 and CD8 T cells (28–30). Interestingly, IL-2 therapy resulted in expansion and excess activation of CD4 but not CD8 T cells in the current study. Therefore, it is possible that the lack of a positive effect of adjunctive immunotherapy with IL-2 on clinical, bacteriologic, and radiographic responses is due, at least in part, to its inability to elicit CD8 T cell responses necessary for effective host defense against M. tuberculosis. The reasons underlying the differing results between our study and the earlier South African and Bangladesh trials are unclear but may be due to differences in the populations studied and the bacteriologic methods. All the studies involved patients with severe forms of pulmonary TB. The earlier South African and Bangladesh studies were small, and only the South African trial in patients with MDR TB was a randomized trial. The South African and Bangladesh studies relied on sputum smear results, whereas the current trial assessed changes in sputum bacillary load by both qualitative and quantitative cultures performed at frequent intervals. The most notable difference between the earlier and current studies is the inclusion of patients with MDR TB in the South African and Bangladesh trials. MDR TB is more difficult to treat with currently available chemotherapy, and the effect of immunotherapy might be more evident in patients with drug-resistant disease. Nonetheless, despite careful quantitative microbiologic surveillance, we were unable to demonstrate any positive impact of IL-2 immunotherapy on bacillary clearance in patients with advanced cavitary TB. Our study has several important limitations. First, the intradermal IL-2 injections produce recognizable stigmata, such as warmth and pruritus, compared with placebo. Patients and examiners may have been able to determine treatment assignment by inspecting injection site, thus introducing ascertainment bias. Observation bias was minimized during the study by the use of dedicated nurse-injectors, who were not responsible for other patient assessments, to administer the test article. In addition, sputum smears and cultures, immunologic assessments, and chest X-ray interpretations were performed without knowledge of treatment assignment. Second, the trial was a Phase II study focusing on preliminary evidence of microbiologic and immunologic activity of IL-2 immunotherapy in patients in whom drug-susceptible TB was newly diagnosed. Our sample size estimate was calculated to have 80% power to detect a 19% improvement in sputum culture conversion after 1 and 2 months of anti-TB therapy, a difference similar to that of adding rifampin to combination chemotherapy. Our study had lower power to detect smaller improvements in bacteriologic responses to treatment that might be beneficial for some patients. The trial also was not powered to detect significant differences in final TB treatment outcomes such as relapse between the immunotherapy and control arms. Finally, we analyzed patients with drug-susceptible and INH-monoresistant TB and cannot, therefore, exclude the possibility of an effect in patients with highly drug resistant TB. We studied only one dosing schedule of adjunctive IL-2; however, the regimen used in the current trial was selected on the basis of positive results with this regimen in published studies and the best information available at the time the trial protocol was designed. Our data from a double-blind, placebo-controlled clinical trial in patients with advanced, drug-susceptible pulmonary TB showed that, despite evidence of a transient systemic effect of adjunctive IL-2, immunotherapy with 450,000 IU of intradermal IL-2 daily during the first month of TB treatment did not enhance bacillary clearance or improvement in symptoms in human immunodeficiency virus–seronegative adults with drug-susceptible TB. Although IL-2 might potentially be of benefit in patients with MDR TB where drug treatment options and responses are sub optimal, our data suggest that adjunctive IL-2 immunotherapy is unlikely to improve results with current rifampin-containing short-course chemotherapy regimens in drug-sensitive TB.
The authors thank the patients and staff of the Ugandan National Tuberculosis Treatment Center, Mulago Hospital; the Ugandan National Tuberculosis and Leprosy Programme; the Uganda Tuberculosis Investigations Bacteriological Unit, Wandegeya, Kampala; and the clinical microbiology laboratories of the Joint Clinical Research Centre, Kampala, Uganda for their invaluable help with the study. Dr. Gilla Kaplan of the Public Health Research Institute, Newark, NJ assisted with study design and protocol development. Sisters C. Drajoru, T. Nakazibwe, and L. Nakalanzi provided outstanding nursing care during the inpatient phase of the study. J. Milman, M.P.H. and M. Millard, M.S.N., M.P.H. provided key on-site project coordination. Dr. Christopher Whalen of the Department of Epidemiology and Biostatistics at Case Western Reserve University supervised the data analysis for the study. The authors also thank Rebecca Elliott and Sach Lai of Chiron for performing the anti–IL-2 antibody assays for the study and Dr. Guido Vanham of the Tropical Medicine Institute of Antwerp for analysis and quality control of the flow cytometric measurements.
Supported by contract NO1-AI45244/AI95383 (Tuberculosis Prevention and Control Research Unit) of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. The recombinant human IL-2 (Proleukin, aldesleukin) used in the study was donated by Chiron Corporation, Emeryville, CA. This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Received in original form November 21, 2002; accepted in final form April 17, 2003
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ABSTRACT
METHODS
= 0.05 [one-tailed]) to detect a minimum difference of 19% in sputum culture conversion rates after 1 and 2 months of anti-TB treatment comparing the IL-2 and placebo groups, assuming a 75% culture conversion rate after 2 months of treatment with standard chemotherapy alone (
