Autocrine Regulation of IL-12 Receptor Expression Is Independent of Secondary IFN- Secretion and not Restricted to T and NK Cells Deborah K. Thibodeaux, Sharon E. Hunter, Kristine E. Waldburger, 1 Judy L. Bliss, William L. Trepicchio, Joseph P. Sypek, Kyriaki Dunussi-Joannopoulos, Samuel J. Goldman, and John P. Leonard 2 The biological response to IL-12 is mediated through specific binding to a high affinity receptor complex composed of at least two subunits (designated IL-12R1 and IL-12R2) that are expressed on NK cells and activated T cells. The selective loss of IL-12R2 expression during Th2 T cell differentiation suggests that regulation of this receptor component may govern IL-12 responsiveness. In murine assays, down-regulation of IL-12R2 expression can be prevented by treatment with IFN-, indicating that receptor expression and hence IL-12 responsiveness may be regulated, at least in part, by the local cytokine milieu. In this study, we report that cellular expression of both IL-12R1 and 2 mRNA is increased in the lymph nodes of naive mice following systemic administration of murine rIL-12 (rmIL-12). Changes in IL-12R mRNA were associated with increased IFN- secretion following ex vivo activation of lymph node cells with rmIL-12, indicating the presence of a functional receptor complex. Expression of IL-12R mRNA was not restricted to lymph node T cells, and its autocrine regulation was independent of secondary IFN- secretion. Data from fractionated lymph node cells as well as rmIL-12-treated B cell-deficient mice suggest that IL-12-responsive B cells may represent an alternative cellular source for IFN- production. However, the strength of the biological response to rmIL-12 is not governed solely by receptor expression, as rmIL-12-induced IFN- secretion from cultured lymph node cells is accessory cell dependent and can be partially blocked by inhibition of B7 costimulation. The Journal of Immunology, 1999, 163: 5257–5264. I nterleukin-12 is a 70-kDa heterodimeric cytokine that is com- prised of disulfide-linked subunits of 35 and 40 kDa, respec- tively. IL-12 is produced primarily by APC following stim- ulation with bacteria, or bacterial products such as LPS (1–3). A T cell-dependent pathway for macrophage and dendritic cell produc- tion of IL-12 following CD40/CD40 ligand activation has also been described (4 –9). In vitro and in vivo studies with the recom- binant and murine proteins have demonstrated broad immunolog- ical activities for IL-12, which have been reviewed in detail else- where (10, 11) The biological response to IL-12 is mediated through specific binding to a high affinity receptor complex that is present on NK cells and activated lymphocytes (12–14). Scatchard analysis of io- dinated rhIL-12 3 binding to human PHA blasts indicates the pres- ence of at least two binding sites with high (5–20 pM) and low (2–6 nM) affinities, respectively (15). Consistent with this obser- vation, cDNAs encoding two distinct IL-12R proteins (designated IL-12R1 and IL-12R2) have been identified by expression clon- ing. IL-12R1 is a member of the gp130 receptor family and binds IL-12 with relatively low affinity. However, IL-12R1 is required for signaling, as mAbs against this subunit inhibit rhIL-12-induced activation (12), and IL-12R1-deficient mice are unresponsive to rmIL-12 (16). A second component of the IL-12R was subse- quently identified (IL-12R2) that by itself binds IL-12 with low affinity, but confers high affinity binding (55 pM) and IL-12 re- sponsiveness when coexpressed in cells with IL-12R1 (15). Reg- ulation of IL-12R expression in the context of Th1 and Th2 dif- ferentiation has been studied in vitro using both murine and human T cells (17, 18). In both instances, Th2 cell development that re- sults in abrogation of IL-12 responsiveness is associated with the specific loss of IL-12R2 mRNA expression. In these in vitro sys- tems, coincubation of murine and human T cells with either IFN- or IFN-/ respectively during Th2 differentiation results in main- tenance of IL-12R2 expression and hence the ability to respond to IL-12 stimulation. Although Abs against IL-12R1 bind to resting PBMCs, acti- vation with anti-CD3 Ab or PHA is required to promote high af- finity binding of labeled rhIL-12 (12) and responsiveness to IL-12 stimulation (19, 20). Despite the presence of the IL-12R complex on activated T cells, depletion of monocytes from PHA blasts par- tially abrogates the ability of IL-12 to induce IFN-. These data suggest that expression of the IL-12R complex alone is not suffi- cient to confer maximum biological responsiveness to IL-12. Fur- ther analysis demonstrated that this monocyte-dependent T cell response to IL-12 stimulation was mediated predominantly via the CD58/CD2 interaction (21, 22). The observation that T cell re- sponsiveness to IL-12 is dependent on signals provided by acces- sory cells is supported further by reports demonstrating synergy between IL-12 and B7 for IFN- secretion (23, 24). Taken to- gether, these observations indicate that expression of both chains Preclinical Research and Development, Genetics Institute, Andover, MA 01810 Received for publication April 12, 1999. Accepted for publication August 31, 1999. 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 Current address: Merck Research Laboratories, Rahway, NJ 07065. 2 Address correspondence and reprint requests to Dr. J. P. Leonard, Genetics Institute, Preclinical R&D, One Burtt Road, Andover, MA 01810. E-mail address: jleonard@genetics.com 3 Abbreviations used in this paper: rh, recombinant human; LNC, lymph node cell; rm, recombinant murine. Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00