Novel immune deficiencies: defective transcription of lymphokine genes

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Novel immune deficiencies: defective transcription of lymphokine genes RAIF S. GEHA, MD, EMANUELA CASTIGLI, MD and TALAL CHATILA, MD Division of Immunology, Children's- Hospital, and the Department of Pediatrics, Harvard Medical School, Boston, MA

Introduction: Cellular and humoral immune responses to antigens are strictly dependent on the synthesis by activated T cells of a multitude of soluble factors. These factors include the interleukins, interferon-y, colony stimulating factors, tumor necrosis factor a and p, transforming growth factor p, and other yet uncharacterized factors. [1,2] We describe a case of primary severe combined immunodeficiency (SCID) with normal number and phenotype of circulating lymphocytes which were deficient in their capacity to synthesize multiple lymphokines. Case Report: The patient is a 3 years and eight months old girl born at 3 7 weeks of gestation. The patient developed, at 2 months of age, a generalized vesicular eruption after exposure to a sibling with chicken pox. This ultimately resolved following intravenous therapy with acyclovir. Investigation of her immune function at six months of age revealed normal lymphocyte count and phenotype, absent delayed type hypersensitivity response to a battery of seven antigens, impaired in vitro proliferation of T-cells to mitogens, hypogammaglobulinemia (IgG 170 mg/ml; IgA < lmg/ml; IgM 12 mg/ml), and low varicella zoster titers (IgG titerl:16; IgM titer< 1:8). Tests for HIV in the child and in the mother were negative. Tests for the activity of adenosine deaminase and purine nucleoside phosphorylase were normal. The patient was placed on intravenous gamma globulin yet she suffered from progressive opportunistic infections including oral thrush and Pneumocystis carinii pneumonia, and failed to thrive. Two bone marrow transplants at 6 and 8 months of age were transplanted with a T cell depleted haploidentical (paternal) bone marrow graft. Based on the observation that the patients T cells failed to secrete detectable amounts of IL-2 upon stimulation with mitogens but proliferated well to the combination of mitogens and exogenous IL-2, the patient was started on intravenous IL-2 replacement therapy with a remarkable clinical response. [3]

Proceedings of EAACI 1990 Meeting

Results Flow cytometric analysis of the patient's circulating lymphocytes at the age of 6 months revealed normal absolute numbers and percentages of T cells that expressed the T cell receptor/CD3 complex, CD4 and CD8. Despite their normal number and phenotype, the patient's lymphocytes failed to proliferate in response to the mitogenic anti CD3 monoclonal antibody 0KT3 to a mitogenic combination of anti CD2 monoclonal antibodies, OKTU + 9.6 or to Phytohemaglutinin. [Table 1] Table 1 demonstrates that the various mitogens tested induced the expression of IL-2 receptor on the patient's T cells but to a lesser extent than that observed for control T cells. The secretion of IL-2 and of interferon-y by the patient's T cells upon their activation by lectins or by agonistic antibodies was either severely defective or undetectable. In contrast, monocyte derived production of IL-6 and TNFa was normal. The impaired proliferation of the patient's T cells to mitogens was dramatically enhanced by IL2. [Table 1] The defective synthesis of multiple lymphokines by the patient's T cells could be the result of an abnormality affecting the transduction via cell surface receptors of signals necessary for the induction of lymphokine synthesis e.g. elevation in free intracellular Ca+-f [Ca++]i, and activation of protein kinase C (PKC). Alternatively the defective production of lymphokines by the patient's lymphocytes could have resulted from a more distal abnormality. Treatment of the patient's peripheral blood lymphocytes with the PKC activator, PMA and the calcium ionophore ionomycin could induce only a modest synthesis of both IL-2 interferon-y and IL-4. This suggested that the defective synthesis of lymphokines by the patient's peripheral blood T cells was related to an abnormality distal to the generation of second messengers which follows the engagement of the T cell receptor/CD3 complex [4]. T cell lines were generated from the patient's peripheral lymphocytes and were similar to the patient's peripheral blood T cells in their inability to secrete lymphokines upon stimulation with mitogens. Northern blot analysis of total cellular RNA isolated from these cell lines upon activation with PMA and ionomycin revealed profound decrease in the levels of mRNA coding for IL-2, IL-3, IL-4, IL5, and interferon-y with otherwise normal message size for each of these lymphokines [Figurel]. The time course for lymphokine mRNA accumulation in patient and in control lymphocytes was identical. The specificity of these abnormalities was suggested by normal mRNA levels found for two other genes transcribed by T cells, the cytokine GM-CSF [Figure 1] ahd the T cell receptor-associated protein CD3y . The \decreased. lymphokine mRNA levels could result from a decrea:sed' rate of initiation of lymphokine /gene transcription upon lymphocyte activation. Alternatively, it could result from enhanced degredation of lymphokine mRNA which are otherwise transcribed at a

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normal rate. To differentiate between these 2 possibilities, we used a nuclear runoff transcription assay. - The results obtained showed that the rate of initiation of IL-2, IL-4 and IL-5 transcription upon cell activation was markedly decreased in the patient's T cells as compared to control lymphocytes (data not shown). Thus the defect in the patients lymphocytes involved a failure of initiation of lymphokine gene transcription. The defect in the patients lymphocytes likely involves an abnormality affecting a regulatory factor that participates in the initiation of transcription of these lymphokines upon T cell activation. The causal relationship between defective IL-2 production by the patient's lymphocytes and her immunodeficiency state was highlighted by the complete correction of the poor jji vitro proliferative responses of her lymphocytes to mitogens and the improvement in her clinical status upon initiation of IL-2 replacement therapy [3]. Interestingly, despite the defective expression of multiple lymphokines, replacement therapy with IL-2 alone was sufficient to restore her immune function. IL-2 could upregulate the production of some lymphokines that are defectively expressed by the patient's lymphocytes e.g.interferon-y. Indeed IL-2 supplementation caused a modest increase in interferon-y production by the patient's lymphocytes (data not shown). The function of other lymphokines such as lL-3 is to a large extent redundant with that of cytokines such as colony stimulating factors (e.g. GM-CSF), the production of which was apparently unaffected. This would account for the normal hematopoiesis observed in this patient.

Proceedings of EA A CI 1990 Meeting

TABLE 1. IMMUNOLOGIC FUNCTION OF PATIENT AND CONTROL T LYMPHOCYTES A.

Proliferative responses to mitogens*

Stimulus

Medium Medium-i-IL-2 Anti-CD3 (0KT3) Anti CD 3+IL-2 Anti-CD2 Anti-CD2+IL-2 PHA PHA+IL-2

3H cpm Thymidine Incorporation Patient

Control

538 13336 720 67785 673 298610 25583 521010

1911 60,060 22680 198090 183487 268160 365690 418070

B. Lymphokine and monokine synthesis following PMA plus ionomycin stimulation.** Patient IL-2 (Femtomoles/ml) IFN- Y(U/ml) IL-4 (pg/ml) IL-6 (ng/ml) TNFa (pg/ml)

69 75 3 29 400

Control 2745 782 110 30 370

*Recombinant IL-2 was used at 100 U/ml, all monoclonal antibodies were used at 1 fig/ml, and PHA-P was used at 10 |j,g/ml. Results shown are mean of triplicate determinations. Similar results were found in 5 other experiments. **PMA was used at 20 ng/ml and ionomycin at 0.5|j,M. Similar results were found in 3 other experiments. N.D: not detectable.

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Pahwa R, Chatila T, Pahwa S, Paradise C, Day NK, Geha RS, Schwartz SA, Slade H, Oyaizu N, Good RA. Recombinant interleukin 2 therapy in severe combined immunodificiency. Proc Natl Acad Sci USA 1989; 86; 5069-73.

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Weiss A, Imboden J, Hardy K, Manger B, Terhorst C, Stobo J. The role of the antigen receptor/T3 complex in T-cell activation. Annu Rev Immunol 1986; 4; 539-619.

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Chatila T, Wong R, Young U, Miller R, Terhorst C, Geha RS. An Immunodeficiency characterized by defective signal transduction in T lymphocytes. N Engl J Med 1989; 320; 696-701.