Allergen-Induced Airway Hyperreactivity Is Diminished in CD81-Deficient Mice 1 Jun Deng,* V. Pete Yeung, ² Daphne Tsitoura, ² Rosemarie H. DeKruyff, ² Dale T. Umetsu, ² and Shoshana Levy 2 * We demonstrated previously that CD81 2/2 mice have an impaired Th2 response. To determine whether this impairment affected allergen-induced airway hyperreactivity (AHR), CD81 2/2 BALB/c mice and CD81 1/1 littermates were sensitized i.p. and chal- lenged intranasally with OVA. Although wild type developed severe AHR, CD81 2/2 mice showed normal airway reactivity and reduced airway inflammation. Nevertheless, OVA-specific T cell proliferation was similar in both groups of mice. Analysis of cytokines secreted by the responding CD81 2/2 T cells, particularly those derived from peribronchial draining lymph nodes, revealed a dramatic reduction in IL-4, IL-5, and IL-13 synthesis. The decrease in cytokine production was not due to an intrinsic T cell deficiency because naive CD81 2/2 T cells responded to polyclonal Th1 and Th2 stimulation with normal proliferation and cytokine production. Moreover, there was an increase in T cells and a decrease in B cells in peribronchial lymph nodes and in spleens of immunized CD81 2/2 mice compared with wild-type animals. Interestingly, OVA-specific Ig levels, including IgE, were similar in CD81 2/2 and CD81 1/1 mice. Thus, CD81 plays a role in the development of AHR not by influencing Ag-specific IgE production but by regulating local cytokine production. The Journal of Immunology, 2000, 165: 5054 –5061. A sthma is a chronic airway disease characterized by in- flammation, airway hyperreactivity (AHR), 3 and reversible airway obstruction (1). The lung inflammatory responses in asthma are tightly associated with airway hyperresponsiveness, and Th2 cells play a critical role in initiating and sustaining asthmatic lung inflammation. In asthma patients, activated CD4 1 T cells with predominant Th2 type cytokine (IL-4 and IL-5) expression are present in bronchoalveolar lavage (BAL) fluid and in lung biopsies (2–5). In murine models, the importance of CD4 1 Th2 cells in the development of pulmonary allergic inflammation has also been demonstrated. Adoptive transfer of Ag-specific Th2 cells into na- ive mice induced AHR and lung inflammation upon Ag exposure (6 – 8). Absent or reduced levels of IL-4 in IL-4 2/2 mice or in mice treated with anti-IL-4 Ab inhibited both airway eosinophilia and AHR (9, 10). Mice lacking the STAT6 transcriptional factor, im- portant in mediating IL-4-downstream events (11, 12), are pro- tected from allergen-induced bronchial eosinophilia, inflammation, and AHR (13, 14). In addition, neutralization of IL-13, another Th2 cytokine, with the soluble IL-13 receptor a 2 , significantly at- tenuated the asthma phenotype in OVA-sensitized and -challenged BALB/c mice (15, 16). We reasoned that because Th2 cells are necessary and sufficient for the induction of AHR, factors affecting Th2 immune responses might influence the development of asthma. One such factor is the CD81 tetraspanin protein. CD81 (TAPA-1, the target of an anti-proliferative Ab) is a widely expressed cell surface protein involved in a variety of bi- ological responses that has been studied mostly in the context of the immune system (17). On T cells, it associates with CD4 and CD8 and was shown to be involved in T cell differentiation (18– 20). On B cells, it associates in a B cell-specific complex with CD19, CD21, and the IFN-inducible Ag Leu13 (21–23), with MHC class II molecules (24, 25), with integrins, and with other tetraspanins (26, 27). Functional studies suggest that CD81 is in- volved in cell motility, adhesion, proliferation, and differentiation (17). CD81-deficient (CD81 2/2 ) mice have an impaired humoral immune responses to protein Ags (28 –30). Chimeric mice, in which only the B cells lacked CD81, were also deficient in Th2 responses as evidenced by their reduced production of Ag-specific IgG1 Ab and IL-4 (31). The goal of this project was to study the role of CD81 in the context of a physiological Th2-dependent response. For this pur- pose, we compared the effect of allergen exposure on the devel- opment of AHR, airway eosinophil inflammation, and cellular and humoral immune responses in CD81 2/2 mice and their wild-type littermates. Our results indicate that expression of CD81 is essen- tial for the development of AHR and for local cytokine production although it has no effects on T cell proliferation or on specific IgE response to the allergen. Materials and Methods Animals CD81 2/2 mice were generated as described (28) and backcrossed six times to BALB/c mice, obtained from Stanford Medical Center Division of Lab- oratory Animal Medicine (Stanford, CA). After the fourth backcross, CD81 2/2 mice could no longer reproduce and had to be maintained as heterozygous animals. Heterozygous mice from the sixth backcrosses were crossed once more to BALB/cByJ mice obtained from The Jackson Lab- oratory (Bar Harbor, ME). Heterozygous mice from the seventh backcross were bred to produce CD81 1/1 and CD81 2/2 littermates. *Division of Oncology, Department of Medicine, and ² Division of Immunology and Transplantation Biology, Department of Pediatrics, Stanford University Medical Cen- ter, Stanford, CA 94305 Received for publication May 23, 2000. Accepted for publication July 31, 2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grants CA34233, AI37219, and AI45900. J.D. was supported by National Institutes of Health Training Grant 5 T32 AI07290-15, “Molecular and Cellular Immunobiology.” 2 Address correspondence and reprint requests to Dr. Shoshana Levy, Department of Medicine/Oncology, Room 1105a CCSR, Stanford University Medical Center, Stan- ford, CA 94305. E-mail address: levy@cmgm.stanford.edu 3 Abbreviations used in this paper: AHR, airway hyperreactivity; BAL, bronchoal- veolar lavage; LN, lymph node; PAS, periodic acid-Schiff. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00