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Helper T lymphocytes (CD4+ T cells) play a central part in normal and pathologic immune responses through the secretion of cytokines. Interleukin-2, interferon gamma, and tumor necrosis factor are involved in cell-mediated immunity, whereas interleukin-4 stimulates the production of IgE antibodies and interleukin-5 promotes the differentiation and activation of eosinophils4,5. Functional analysis of murine and human T-cell clones generated in vitro led to the identification of several subpopulations of CD4+ cells with two strongly polarized subgroups: the type 1 helper T cells, which produce interleukin-2 and interferon gamma but not interleukin-4 or interleukin-5, and type 2 helper T cells, which produce interleukin-4 and interleukin-5 but not interleukin-2 or interferon gamma6,7. These two subgroups may have a role in several immunopathologic processes. Indeed, cells resembling type 1 helper T cells have been found in infectious granulomatous diseases, whereas cells resembling type 2 helper T cells have been identified in lepromatous leprosy, visceral leishmaniasis, and atopic disorders8,9,10,11,12,13. However, studies of monoclonal T-cell disorders are required to determine whether clones of human type 2 helper T cells arise in vivo. We investigated this possibility in a man with the hypereosinophilic syndrome characterized by excessive production of serum IgE and clonal expansion of CD4+CD3- T cells. We found that this T-cell clone produced high levels of interleukin-4 and interleukin-5 but had a markedly reduced ability to secrete interleukin-2 and interferon gamma. This observation indicates that clones of type 2 helper T cells differentiate in vivo and suggests that clonal expansion of type 2 helper T cells can cause the hypereosinophilic syndrome.
Case Report
A 30-year-old man presented with a four-month history of generalized pruritus, a cough productive of yellowish sputum, intermittent fever, and exertional dyspnea. Physical examination disclosed a few papular skin lesions and some bronchial rales. Major laboratory findings included marked eosinophilia (absolute eosinophil count, 6117 per cubic millimeter) and a polyclonal increase in serum levels of IgM (7200 mg per deciliter; normal, <250) and IgE (2000 IU per milliliter [4800 µg per liter]; normal, <100 IU per milliliter [240 µg per liter]). Immunophenotyping of peripheral-blood mononuclear cells (PBMC) by flow cytometry revealed the following proportions of cells: 42 percent CD3+, 75 percent CD4+, 16 percent CD8+, and 90 percent CD2+ T cells. Double- and triple-staining studies indicated that 66 percent of the CD4+ cells did not express CD3 on their surface. These CD4+CD3- cells did not stain with monoclonal antibodies against 
/(
) or 
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T-cell receptors but expressed the CD2 marker. Antibodies were not detected against the human immunodeficiency virus types 1 and 2 or human T-cell lymphotropic virus type I. The patient's karyotype was normal. Computed tomography of the thorax revealed slight pleural effusions. Abdominal computed tomography and echocardiography revealed no abnormalities. A skin biopsy demonstrated a dermal perivascular infiltration with monocytes and numerous eosinophils. Oral administration of methylprednisolone was begun at a dose of 32 mg per day and resulted in rapid clinical improvement and a drop in the eosinophil count (Figure 1) and in serum IgM and IgE levels (data not shown). When the dose of methylprednisolone was tapered, the initial symptoms and biologic abnormalities recurred. In addition, pain and blanching of the first two fingers of the right hand developed. Thrombotic vasculitis was confirmed by a biopsy. An iliac-bone biopsy and histologic examination of a left cervical lymph node revealed no abnormalities except for the presence of numerous eosinophils. An increase in the dose of methylprednisolone to 48 mg per day controlled this flare, but the symptoms recurred when the dose was tapered (Figure 1). Since interferon alfa therapy has been successful in the hypereosinophilic syndrome,14,15,16 a therapeutic trial of subcutaneous interferon alfa-2b (Schering-Plough, Innishannon, Ireland) was started at a dose of 5 million IU per day. The addition of this drug resulted in a rapid decrease in eosinophilia (Figure 1). Serum levels of IgE remained unchanged, whereas serum levels of IgM progressively decreased from 7950 mg per deciliter at the beginning of treatment to 2530 mg per deciliter after two months of interferon alfa-2b. During treatment, the percentage of CD4+CD3- cells decreased from 60 to 31 percent while the percentage of CD3+CD4+ cells increased from 17 to 41 percent.
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Isolation of PBMC
PBMC from the patient and five healthy volunteers were isolated from freshly drawn heparin-treated blood by density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway). CD4+ cells were selected through the use of immunomagnetic beads coated with an anti-CD4 monoclonal antibody (Dynabeads M450 CD4 and Detachabead, Dynal, Oslo, Norway). Non-T cells were obtained by the successive depletion of CD4+ cells and CD8+ cells with the use of immunomagnetic beads coated with corresponding monoclonal antibodies (Immunotech, Marseilles, France). The patient's CD4+CD3- cells were prepared from CD4+ cells by the selective depletion of CD3+ cells through incubation with an anti-CD3 monoclonal antibody (Ortho Biotech, Raritan, N.J.) and immunomagnetic particles coated with goat antimouse IgG (Immunotech). The purity of the resulting cell preparations was more than 95 percent, as determined by flow cytometry.
Southern Blot Analysis of Gene Coding for the Chain of the T-Cell Receptor
Southern blot analysis of the gene coding for the chain of the T-cell receptor was performed according to standard procedures17,18. The probe (JUR-2) used was a complementary DNA clone of the second constant region of the gene19.
Cell Cultures
Purified CD4+ or CD4+CD3- cells were cultured at a density of 1 x 106 per milliliter in RPMI-1640 medium containing 10 percent fetal-calf serum (Myoclone, GIBCO, Life Technologies, Paisley, Scotland). In some experiments, a combination of 0.1 µg of A23187 calcium ionophore per milliliter (Calbiochem-Behring, San Diego, Calif.) and 10 ng of phorbol myristate acetate per milliliter (Sigma Chemical, St. Louis) was added to the culture medium as a stimulating agent. After 48 hours of incubation at 37 °C in an atmosphere of 5 percent carbon dioxide, the supernatants were collected and stored at -20 °C until assayed for cytokines.
Determination of Cytokine Levels in Serum and Culture Supernatants
Levels of interleukin-5 in serum and culture supernatants were determined by enzyme-linked immunosorbent assay as previously described20. Commercially available kits for enzyme-linked immunosorbent assay were used to determine interleukin-2 levels (Medgenix, Fleurus, Belgium) and interleukin-4 levels (Quantikine R and D Systems, Minneapolis). Levels of interferon gamma were measured by an immunoradiometric assay (Medgenix).
Results
Analysis of Rearrangements of the Chain of the T-Cell-Receptor Gene
Southern blot analysis of the DNA extracted from the patient's PBMC demonstrated the presence of a monoclonal-cell population in which the two alleles of the chain of the T-cell-receptor gene were rearranged. The rearrangement was characterized by two bands localized on both sides of the 9.0-kb germline band (Figure 2). The analysis of whole PBMC was repeated with three different restriction enzymes; all analyses demonstrated a clear pattern of clonal rearrangement, whereas no clonality was observed in a control patient with the hypereosinophilic syndrome who had a normal T-cell phenotype (data not shown). Analysis of purified cell populations revealed that the rearrangement pattern was present only in CD4+CD3- cells, whereas only the normal germline pattern was observed in CD4+CD3+ cells as well as in non-T cells (Figure 2). We concluded that the abnormal CD4+CD3- T-cell population was monoclonal and that the T-cell monoclonality was restricted to this population.
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First, purified CD4+ cells from the patient and five normal subjects were cultured in the absence of any stimulating agent. As shown in Table 1, the patient's CD4+ cells spontaneously produced measurable levels of interleukin-4 and interleukin-5, whereas levels of interleukin-2 and interferon gamma were undetectable. The CD4+ cells isolated from normal subjects did not produce any detectable cytokine. The pattern of cytokine secretion by the patient's CD4+CD3- cells established the role of this monoclonal T-cell population in the spontaneous production of interleukin-4 and interleukin-5 (Table 1).
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Serum levels of interleukin-5 were monitored to assess the in vivo activity of the type 2 helper T-cell clone (Figure 1). Serum levels were very high before treatment but decreased rapidly after the initiation of therapy with methylprednisolone. This effect of steroids appeared to decrease with time, however. When interferon alfa-2b was added to the regimen, there was a delayed and moderate decrease in interleukin-5 levels.
Discussion
Our patient met the criteria for the hypereosinophilic syndrome, since his condition was characterized by prolonged hypereosinophilia, eosinophilic infiltration of the skin and marrow, and pleuropulmonary manifestations1. As reported in other cases of the syndrome,21,22 the hypereosinophilia was clearly associated with the production of interleukin-5, a cytokine known to mediate eosinophilia5. In our patient, interleukin-5 was secreted by a phenotypically abnormal CD4+CD3- cell population that proved to be monoclonal on analysis of the chain of the T-cell-receptor gene. In addition to producing large amounts of interleukin-5, this T-cell clone also produced high levels of interleukin-4 but only very low or undetectable levels of interleukin-2 and interferon gamma. This pattern of cytokine production corresponds to the definition of type 2 helper T cells6. Our findings therefore establish that a clonal expansion of type 2 helper T cells does occur in humans. The pathologic features of this clonal disease appear to be directly linked to the production of cytokines, with interleukin-5 being responsible for the hypereosinophilia5 and interleukin-4 for excessive production of IgE4.
The CD4+CD3- phenotype of the monoclonal T cells has previously been observed in patients with lymphoma associated with the hypereosinophilic syndrome23,24 and in a patient with T-cell lymphoma presenting as the hypereosinophilic syndrome and vasculitis2. This phenotype might be due to the deficient synthesis of one or several of the polypeptide chains that constitute the complex formed by T-cell receptors and CD3+ cells. As with other "benign" diseases mediated by clonal T cells,18 clonal expansion of type 2 helper T cells associated with hypereosinophilia may represent a premalignant condition. A pattern of cytokine secretion resembling that of type 2 helper T cells has recently been demonstrated in Sezary syndrome, a CD4+ lymphoproliferative disorder associated with hypereosinophilia and excessive serum levels of IgE25.
Since serum levels of interleukin-5 dropped dramatically with steroid treatment, the initial response was probably mediated by the inhibition of interleukin-5 production by type 2 helper T cells as well as by a direct effect on the eosinophils26. On the other hand, the reduction in the eosinophil count after the initiation of interferon alfa-2b therapy could be related to a direct inhibition of the differentiation or proliferation of eosinophils27.
Our observations emphasize that patients with the hypereosinophilic syndrome should be carefully examined for an underlying proliferation of clonal T cells. Future studies should determine the best way to prevent this type of hypereosinophilic syndrome from becoming a full-blown malignant lymphoproliferative disorder.
Supported by grants from the Fonds de la Recherche Scientifique Medicale (Belgium) and the Universite Libre de Bruxelles.
We are indebted to Dr. L.A. McNamee (Glaxo, Greenford, United Kingdom) and Dr. R. Devos (Roche Research, Ghent, Belgium) for providing the anti-interleukin-5 antibodies used to measure interleukin-5, to Dr. C. Bruyns for helping with cell cultures, and to Schering-Plough, Belgium, for supplying interferon alfa-2b (Intron A).
Source Information
From the Department of Internal Medicine, Hopital Universitaire Brugmann (E.C.), the Department of Immunology, Hopital Universitaire Erasme (L.S., A.C., M.G.), and the Department of Medical Genetics, Institute of Interdisciplinary Research, Universite Libre de Bruxelles (P.C., T.V.) -- all in Brussels, Belgium.
Address reprint requests to Dr. Cogan at the Department of Internal Medicine, Hopital Universitaire Brugmann, 4, place Van Gehuchten, B 1020 Brussels, Belgium.
References
-chain genes. Nature 1984;312:521-524. [CrossRef][Medline]
interferons on human multipotential (CFU-GEMM), erythroid (BFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells. J Immunol 1983;131:1300-1305. [Abstract]
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