cAMP-mediated suppression of a Th1 clone associated with an alteration of the intracellular redox environment.

We have shown previously that the elevation of intracellular cAMP in antigen or anti-CD3-activated murine Th1 clones in the absence of antigen inhibits antigen-induced proliferation and the production of IL-2 by H2O2-mediated oxidation of p56lck and inhibits antigen-induced production of interferon-gamma by the induction of intracellular nitric oxide. Moreover, activated Th1 clones are resistant to cAMP-induced suppression. These results suggest that the immunosuppression of Th1 cells mediated by elevated intracellular cAMP is associated with an alteration in the intracellular oxidation/reduction environment. Here we report that the culture of an antigen or anti-CD3-activated murine Th1 clone with the adenylcyclase agonist forskolin (FSK) in the absence of antigen reduces the activity of intracellular catalase, and diminishes levels of intracellular reduced glutathione (GSH). Resting cells resistant to cAMP-induced suppression have higher intracellular GSH levels than antigen-activated cells susceptible to cAMP-induced suppression. The results provide further evidence that cAMP-induced suppression of Th1 clones is mediated by profound alterations in the intracellular redox environment and may be used to selectively inactivate Th1 cells activated by antigen.

Additional evidence that the sympathetic nervous system regulates the vessel wall release of tissue plasminogen activator.

It is established that sympathetic neurons can synthesize, transport and store tissue plasminogen activator (t-PA) within axon terminals in the smooth muscle of vessel walls. Moreover, sympathetic excitations (e.g. physical and mental stress) are known to induce an acute release of t-PA into the circulation. However, relatively little is known about the nature and extent of sympathetic nervous system involvement in the release process. We inquired whether a chemical sympathectomy will alter the release of t-PA into the blood, and the intrinsic release of stored t-PA from isolated whole vessel explants. A long-term sympathectomy was induced in adult Sprague-Dawley rats by injection of guanethidine during a 5-week course. The destruction of ganglion neurons and vessel wall axons was verified immunohistochemically. t-PA release was assayed as the free activity in hind limb plasma and explant culture medium. Following sympathectomy: (i) the basal t-PA activity in plasma was 70% less than controls (2.92 +/- 1.96 versus 9.33 +/- 1.72 IU/ml;

Differential regulation of T cell receptor-mediated Th1 cell IFN-gamma production and proliferation by divergent cAMP-mediated redox pathways.

Culture of an H-2(s)-restricted, bovine myelin basic protein (BMBP)-specific murine Th1 clone with the adenyl cyclase agonist forskolin (FSK) or isobutylmethylxanthine (IBMX), an inhibitor of cAMP catabolism, before culture with anti-CD3 or BMBP and antigen-presenting cells (APC) suppressed antigen or anti-CD3-induced proliferation and production of interferon-gamma (IFN-gamma). Other H-2(s)-derived or H-2(b)-derived clones specific for BMBP or keyhole limpet hemocyanin (KLH) were similarly affected. FSK did not affect the expression of CD4 or the T cell receptor (TCR) but did diminish levels of the phosphorylated (activated) mitogen-activated protein (MAP) kinases early response kinase-1 (ERK-1) and ERK-2. Immunoblotting of lysates from an FSK-treated Th1 clone with antibodies to a carboxy-terminal epitope of p56(lck), a signal transduction enzyme upstream from ERK-1 and ERK2, did not detect p56(lck) unless the lysates were reduced prior to electrophoresis. Immunoblotting of nonreduced lysates with antibodies to an amino-terminal epitope demonstrated p56(lck) with a lower apparent molecular weight, characteristic of oxidized proteins. Reduction restored the detection of p56(lck) by anticarboxy-terminal p56(lck) and to mobilities indistinguishable from controls detected by the antiamino-terminal p56(lck). N-acetylcysteine or catalase prevented FSK-induced suppression of antigen-induced proliferation and the loss of carboxy-terminal epitopes of p56(lck). An inhibitor of cAMP-dependent protein kinase A (PKA) or nitric oxide synthase (NOS) did not affect FSK-induced inhibition of antigen-induced proliferation. In contrast, inhibitors of PKA or NOS, but not catalase, prevented FSK-induced suppression of IFN-gamma production. Moreover, immunoblots of lysates precipitated with anti-p56(lck), phosphotyrosine, or CD4 demonstrated that in FSK-treated, anti-CD3-stimulated cells, p56(lck) is not associated with CD4 zeta chain, nor is p56(lck) or zeta chain phosphorylated. In vitro kinase assays demonstrated that p56(lck) from FSK-treated cells does not have kinase activity. Taken together, the results suggest that an elevation of intracellular cAMP (in the absence of antigen) creates an oxidative environment that oxidizes and inactivates p56(lck) by an H(2)O(2)-dependent, PKA-independent mechanism and inhibits the production of IFN-gamma by an NO, PKA-dependent mechanism. Thus, antigen-induced proliferation and IFN-gamma production in a Th1 clone are controlled separately by different cAMP-dependent, redox-based mechanisms.

Blood mononuclear cells induce regulatory NK T thymocytes in anterior chamber-associated immune deviation.

Injection of antigen into the anterior chamber (AC) of the eye, an immunologically privileged site, is associated with the induction of immune deviation, as evidenced by T helper cell (Th) 1 to Th2 cell polarization. We recently demonstrated that AC-associated immune deviation (ACAID) is a thymus-dependent phenomenon initiated by the formation of regulatory alpha,beta T-cell receptor-positive CD4(-) CD8(-) thymocytes (THYregs). In this study, the afferent and efferent limbs of this immunoregulatory loop were traced from peripheral blood to the thymus and then to the spleen by adoptive-transfer assays. The results demonstrate that (1) F4/80(+) CD1(+) peripheral blood mononuclear cells from mice whose ACs were injected with trinitrophenol-bovine serum albumin induce the appearance of natural killer (NK) 1.1(+) THYreg in naïve recipients within 24 h of intravenous infusion; (2) these NK THYregs induce (or generate) suppressor-effector T cells in the spleens of adoptive recipients; (3) these suppressor-effector spleen cells, but not the NK THYregs themselves, directly inhibit the expression of delayed-type hypersensitivity in sensitized recipients; and (4) peripheral blood mononuclear cells from AC-injected mice do not induce ACAID in thymectomized recipients. These results confirm our hypothesis that ACAID is a model of centrally induced dominant tolerance mediated by CD-1-dependent NK T cells of recent thymic origin. The results also provide evidence of a novel tolerance induction pathway by which blood-borne antigen-presenting cells generated by antigen injection into an immunologically privileged site transport antigen to the thymus and induce the formation and export of THYreg.

Immunological and in-vivo neurological studies on a benzoic acid-specific T cell-derived antigen-binding molecule from the serum of a toluene-sensitive patient.

T-cell-derived antigen-binding molecules (TABMs) specific for benzoic acid were isolated from the serum of a toluene-sensitive patient. The resulting purified TABMs (BA-TABMs) did not contain immunoglobulin G and were associated with the cytokine transforming growth factor-beta (TGF-beta). BA-TABMs bound to benzoic acid conjugated to human serum albumin (BA-HSA), as well as to other chemicals conjugated to human serum albumin-including dinitrophenol and oxazolone. The binding of BA-TABMs to the conjugated chemicals increased the level of detectable TGF-beta, and a similar effect was observed with the unconjugated chemicals, benzoic acid and 2,4-dinitrophenol glycine. The increase in TGF-beta was critically dependent on the ratio between BA-TABMs and the conjugated or unconjugated chemicals; the increase was optimum at intermediate concentrations and absent at low and high concentrations. The authors used an established animal model in vivo and demonstrated that TGF-beta enhanced the inflammatory response induced by the release of neuropeptides from sensory nerves; this enhancement occurred in a dose-dependent manner. The BA-TABMs also enhanced this neurogenic inflammatory response in a dose-dependent manner, and this effect was blocked by anti-TGF-beta antibody. When the authors added either BA-HSA or benzoic acid, the effect of BA-TABMs on neurogenic inflammation was further enhanced at intermediate concentrations of antigen and was unaltered or reduced at higher concentrations. TABMs specific to particular chemicals, as a result of their association with cytokines (e.g., TGF-beta), may be implicated in symptom production in chemically sensitive patients.

Detection of antigen-specific human serum proteins related to the T-cell receptor in infectious disease and in an immune response to milk proteins or chemicals.

A monoclonal IgG2 antibody, MG3C9-1 A12, was prepared by immunization of mice with human serum Cohn Fraction III proteins enriched for TCR Ca+ proteins. MG3C9-1 A12 bound to Mr 28,000, antigen-specific TCR Ca+, beta-, and TCR Ca+, beta+ serum proteins associated with TGF-beta1, 2. The IgG2 monoclonal antibody also bound to T-lymphocyte proteins but did not bind to B lymphocyte proteins, human albumin, IgM, IgG, IgA, or TGF-beta1, 2, 3 immunogenic peptides. Monoclonal MG3C9-1 A12 detected TCR-related proteins specific for filarial extract, milk proteins, or benzoic acid in the sera of individuals with chronic or asymptomatic filariasis, milk intolerance, or sensitivity to toluene, respectively. TCR-related serum proteins were also detected intracellularly in mononuclear cells in frozen sections of ileum from a patient with milk intolerance and reactive mesenteric lymph nodes from a patient with a gastric ulcer. The results suggest that antigen-specific TCR-related serum proteins may be elevated during an immune response to oral, environmental, or infectious stimuli.

Measurement of T-cell-derived antigen binding molecules and immunoglobulin G specific to Candida albicans mannan in sera of patients with recurrent vulvovaginal candidiasis.

Immunoglobulin G (IgG) and T-cell-derived antigen binding molecules (TABM) specific to whole Candida extract and to Candida-derived mannans prepared by both the cetryltrimethylammonium bromide (CTAB) and alkaline degradation (PEAT) methods were measured in the sera of women with vulvovaginal candidiasis and controls. In the patients there were significantly higher levels of IgG to both CTAB and PEAT mannans and of TABM to CTAB mannan. TABM specific to CTAB mannan was purified from the serum of a patient with a high titer of this TABM. The purified TABM bound specifically to CTAB mannan and to other yeast and mold extracts. This TABM preparation was associated with transforming growth factor beta2 (TGF-beta2), and on specific binding to mannan there was a marked increase in the level of detectable TGF-beta2. This increase in TGF-beta2 level was critically dependent on the relative concentrations of the purified TABM and mannan, being smallest when either was in excess. The TABM specific to CTAB mannan was also shown to inhibit Candida-stimulated gamma interferon production. The results suggest that CTAB mannan-specific TABM may increase susceptibility to vulvovaginal candidiasis in association with a shift in the immune response to the Th2 type.

Sympathectomy decreases and adrenergic stimulation increases the release of tissue plasminogen activator (t-PA) from blood vessels: functional evidence for a neurologic regulation of plasmin production within vessel walls and other tissue matrices.

Our recent morphologic studies indicated that peripheral nervous system (PNS) adrenergic neurons synthesize, transport, and store the serene protease, tissue plasminogen activator (t-PA) in axon terminals, many of which innervate vessel walls. Sympathoadrenal stimulation induces a surge of t-PA from vessel walls into the blood. The vascular endothelium, which constitutively secretes t-PA into blood also has long been widely assumed to be the principal source of this stress-induced release, but has not been verified as such. A neurologically regulated release from adrenergic stores could thus augment the known constitutive endothelial release. To functionally test this possibility, we quantitated the effects of guanethidine-induced systemic sympathectomy on the basal and stimulated release of t-PA from isolated vessel explants in superfused organ cultures. Moment-to-moment changes in the release rate were plotted from serial assays of the t-PA free activity. The effects of endothelial and adventitial nerve plexus ablations were also tested. Sympathectomy induced 30-50% reductions in t-PA release from both arterial and microvascular explants. An acute release induced by alpha-1 adrenergic receptor stimulations was also strongly suppressed, as were basal levels of the circulating enzyme in vivo. Adventitial and endothelial ablations from normal large vessel explants produced greater reductions than small vessel endothelial ablations. Ganglion electrical stimulation also induced an acute microvascular release in vivo. These and past morphologic findings indicate a physiological infusion of t-PA into the vessel walls, blood, and other innervated matrices by sympathetic neurons.

Clinical and immunological responses in subjects sensitive to solvents.

Twenty patients proved sensitive to a 15-min exposure to 15 ppm toluene. We assessed patients neuropsychologically before and after toluene exposure, and they had impaired cognitive functioning characterized by a deterioration in short- and long-term memory and psychomotor coordination. We measured total immunoglobin G and T-cell antigen-binding molecules against an antigen prepared by conjugation of para-aminobenzoic acid to human serum albumin in 20 patients and 16 controls. There was no significant difference in the immunoglobulin G levels to the antigen in the 2 groups, but the levels of T-cell antigen-binding molecules against the para-aminobenzoic acid conjugated to human serum albumin were elevated significantly in subjects sensitive to toluene. We also found significant associations between T-cell antigen-binding molecule levels and (a) decreased performance on the STROOP (Colour Word) test, (b) a shift in focal length following toluene exposure, (c) clinical assessment of disability, and (d) longer histories of chemical exposure. The measurement of T-cell antigen-binding molecules against chemical haptens may be valuable in the assessment of patients who are sensitive to chemicals.

Production of serum immunoglobulins and T cell antigen binding molecules specific for cow's milk antigens in adults intolerant to cow's milk.

The immune response to three cow's milk antigens, beta-lactoglobulin (BLG), alpha-lactalbumin (AL), and casein (CA) was studied in 15 milk-intolerant adult patients and 11 adult controls. IgG, IgE, and IgG subclasses (IgG1, IgG2, IgG3, IgG4) and T cell-derived antigen-binding molecules (TABM) specific for each antigen were measured in both groups. In the patient group, a significant elevation of total IgG and TABM against each of the milk antigens was found as well as raised levels of IgG1 to BLG and CA, IgG4 to BLG, and IgE to CA. TABM specific for BLG were isolated by affinity for BLG and found to be Mr 28,000-46,000 polypeptides functionally and physically associated with TGF-beta1 and TGF-beta2. These results indicate a Th2-type immune response to the milk antigens in milk-intolerant individuals compared with the control group which shows a pattern typical of anergy or deletion.

Functional and morphologic evidence of the presence of tissue-plasminogen activator in vascular nerves: implications for a neurologic control of vessel wall fibrinolysis and rigidity.

Tissue plasminogen activator (t-PA) is expressed by hypothalamic and peripheral sympathetic neurons. The sympathetic axons that permeate artery walls have not been investigated as possible sources of intramural t-PA. The plasmin produced by such a system would locally activate both fibrinolysis and matrix metalloproteinases that regulate arterial collagen turnover. To assess this neural t-PA production, we investigated the capacity of rat cervical sympathetic ganglion neurons to synthesize and release t-PA, and the expression of the enzyme in carotid artery and the iris-choroid microvascular tissues that receive the ganglion axon distribution. Functional studies confirmed that (i) the ganglion neuron cell bodies synthesize t-PA mRNA, (ii) cultured ganglion carotid artery and iris-choroid microvascular explants predominantly release t-PA rather than urokinase, (iii) microvascular tissues release approximately 20 times more t-PA per milligram than carotid explants (which accords with the higher innervation density of small vessels), and (iv) removal of the endothelium did not cause major reductions in the t-PA release from carotid and microvascular explants. Immunolocalization studies then confirmed a strong expression of the enzyme within the ganglion axons, the carotid adventitia that receives these axons, and the predominantly sympathetic axon terminals in the iris-choroid microvasculature. These data indicate the existence of a previously undescribed system for the delivery of neural t-PA to vessel walls. The intramural production of plasmin induced by this system represents a novel principle for the regulation of arterial matrix flexibility, especially in the media of densely innervated small arteries and resistance arterioles involved in the pathogenesis of stroke, hypertension, and vascular aging. Thus, the data suggest an important new interface between neuroscience and vascular biology that merits further exploration.

Extracellular (soluble) antigen-specific T cell proteins related to the T cell receptor for antigen (sTCRr): serologic and primary amino acid sequence similarity to T cell receptor alpha chains and association with cytokines.

Antigen-specific-effected immunoregulation by T lymphocytes is mediated by extracellular proteins produced by T lymphocytes. These immunoproteins bind specifically to nonprocessed antigen and either induce antigen-specific immunoregulatory T cells (tsfi) or effect regulation (tsfe). T cell proteins that bind specifically to nonprocessed antigen have ben termed "T cell antigen-binding molecules" (TABM), and by definition, tsfe and tsfi are, in part, TABM. To characterize tsfi, tsfe, and TABM and understand the relationships and function of these immunoproteins, we have combined the efforts of two laboratories to compare tsfi, tsfe, and TABM isolated by each laboratory. Data obtained in one laboratory were reproduced by the other, and all reagents prepared by each laboratory were exchanged. TABM, tsfi, and tsfe were found to express TCRCalpha epitopes but not TCRCbeta epitopes. The amino acid sequence of a tryptic peptide of a T cell hybridoma TABM specific for nitrophenylhydroxy acetate (NP) is similar to a TCRalpha chain and TCR pre-alpha chain amino acid sequence. ELISA and immunoblotting demonstrated that Mr 77,000 T cell hybrid-derived tsfi, tsfe, and TABM are noncovalently associated with Mr 15,000-16,000 interleukin-10 (IL-10). ELISA also demonstrated that tsfi and tsfe are associated with I-J. The ability of tsfi and tsfe to suppress a mixed lymphocyte reaction was prevented by anti-IL-10 or anti-I-J antibodies, suggesting that antigen-specific immunoregulatory T cell proteins function by an antigen-specific focusing of immunoregulatory cytokines.

Morphologic evidence for a preferential storage of tissue plasminogen activator (t-PA) in perivascular axons of the rat uvea.

The uveal layer is thought to hold the largest stores of tissue plasminogen activator (t-PA) within the eye. However, the uveal cell types that contain and could release t-PA to contiguous tissues and fluids have not been clearly identified. In the present study the general distribution pattern of t-PA antigen in fresh rat iris and choroid tissue was determined by immunofluorescence in preliminary light microscopic (LM) cryosections. Transmission electron microscopic (TEM) immunogold localization was then used to detect specific cellular and subcellular sites of t-PA antigen. The primary antibody was rabbit anti-mouse t-PA IgG. The immunofluorescence in preliminary LM cryosections of both tissues was most intense over discrete linear and cross-sectioned structures that resembled the contours of axon bundles. This impression was strengthened when silver impregnation highlighted similar structures in separate sections of the same tissue samples. TEM immunogold labeling of thin sections then confirmed that the t-PA antigen was confined to the axoplasm of both myelinated and unmyelinated perivascular nerve fibers in both the iris and choroid. Gold particles were not observed over axonal membranes, myelin sheaths, Schwann cells, retinal pigment epithelium or vascular endothelial cells. Ultrathin TEM cryosections of the iris showed a localization of some particles over structures that resembled tubules and vesicles within the axoplasm, but not over mitochondria. The axonal location of t-PA was shown by the co-localization of t-PA with an antibody against rat neurofilaments. The typical axon morphology that enclosed the t-PA particle markers in all TEM sections also indicated an axonal location. Separate TEM sections were processed with conventional fixatives and stains to highlight the typical uveal axon morphology, which also confirmed the identity of perivascular axons as the sites of t-PA localization. Affinity of the primary antibody for rat t-PA was shown by an inhibition ELISA against rat uveal tissue extracts and by the inhibition of t-PA activity in aqueous humor. An amidolytic assay was used to quantify t-PA activity. Possible explanations for the preferential immunolocalization of t-PA antigen to the axoplasm of uveal nerve terminals and the need for additional functional studies to confirm a putative neural t-PA synthesis are discussed.

Direct thymic involvement in anterior chamber-associated immune deviation: evidence for a nondeletional mechanism of centrally induced tolerance to extrathymic antigens in adult mice.

Recent reports have suggested that the dichotomy between central (thymic) and peripheral T cell tolerance is not absolute and that self-tolerance in perinatal animals may also involve the intrathymic generation and release to the periphery of Ag-specific immunoregulatory T cells. We have expanded this concept to include tolerance to non self Ags administered extrathymically to adult animals. In this study, we use the anterior chamber-associated immune deviation (ACAID) to demonstrate that central regulation of acquired peripheral tolerance can be induced in adult mice by the intraocular administration of low doses of nonself Ag. The results show that adult thymectomy prevents the inhibition of trinitrophenol (TNP)-specific delayed-type hypersensitivity, which normally occurs after injection of TNP-BSA into the anterior chamber (AC) of the eye. Thymocytes obtained from mice 1 to 3 days, but not 5 to 7 days, after AC injection of TNP-BSA or BSA alone specifically transfer inhibition of delayed-type hypersensitivity to mice primed with the homologous Ag. The latter observation, when correlated with the time of onset of ACAID, suggests that immunoregulatory T cells are formed in the thymus within 24 h and are exported to the peripheral lymphoid tissues between 2 and 5 days after AC injection of Ag. Immunomagnetic separation of thymocytes revealed that the immunoregulatory activity resides within the minor subset of CD4-, CD8-, TCR-alphabeta+ cells, previously postulated to induce fas ligand-mediated apoptosis and Th1 to Th2 immune deviation. Hence, the present study identifies ACAID as a prototypical model of centrally induced, nondeletional tolerance to extrathymic nonself Ags.

Serum TABM produced during anterior chamber-associated immune deviation passively transfers suppression of delayed-type hypersensitivity to primed mice.

Injection of soluble protein antigen into the anterior chamber of the eye of primed mice induces anterior chamber-associated immune deviation (ACAID) which is manifested by suppression of delayed-type hypersensitivity (DTH) to the antigen. Recently, we found that ACAID induced in primed mice also results in a rapid rise in serum of soluble T lymphocyte-derived proteins specific for nominal antigen (TABM). Here, we demonstrate that serum TABM induced in primed mice during ACAID will transfer the suppression of DTH to mice primed to the same antigen. Sera from TNP-BSA-primed mice that received an anterior chamber injection of TNP-BSA, but not BSA alone, suppressed the DTH response to TNP when injected into other TNP-BSA-primed mice. Sera absorbed with Sepharose beads conjugated with either anti-TCR C(alpha), anti-TCR C(beta), anti-TABM or TNP-BSA did not contain TNP-specific TABM and did not transfer suppression of DTH. These results suggest that the antigen-specific, TCR C(alphabeta)+ TABM that appear in serum during ACAID are able to confer on or amplify the capacity of sensitized T cells to suppress DTH. We believe this to be the first demonstration of an in vivo immunologic function that is specifically associated with TABM produced in vivo.

Induction of Erythropoiesis by MHC-Mediated Cognate Interactions between B- and T-Cells.

We have previously shown that the expression of membrane burst-promoting activity (mBPA), an erythropoietic cytokine, by B-lymphocytes is augmented by the addition of allogeneic effector cells to the B-cells. Here, we have examined immune mechanisms involved in the induction/promotion of erythropoiesis as assessed by the capacity of autologous and allogeneic peripheral blood lymphocytes to augment burst-forming unit-erythroid (BFU-E) in normal human bone marrow cells in vitro. Preincubation of mBPA-expressing human B-cells with monoclonal antibodies to major histocompatibility complex (MHC) antigens, abrogated erythropoietic activity of both autologous and allogeneic lymphocytes, suggesting that MHC antigens play a role in regulating the expression of the erythroid growth factor. Inhibition of BFU-E proliferation was also evident when antibodies to MHC class-I or class-II antigens were added directly to marrow culture. Furthermore, addition of anti-CD4 antibody to the cultures of PBL and autologous target BM cells markedly reduced erythroid proliferation induced by PBL. By contrast, anti-CD8 and control (UPC-10) monoclonal antibodies had no effect. These results provide evidence that MHC-mediated cognate interactions between T- and B-lymphocytes may participate in the control of erythropoiesis, either directly or by modulating mBPA function.

Elevation of intracellular cyclic AMP induces an anergic-like state in Th1 clones.

Because elevated intracellular cAMP suppresses T cell receptor (TCR)-mediated effector activity and/or proliferation in response to antigen but does not always affect IL-2-stimulated proliferation, the effects of cAMP on a T lymphocyte response to antigen resemble antigen-induced anergy. To test the hypothesis that elevated cAMP induces anergy in T lymphocytes, we have precultured murine Th1 clones responsive to porcine myelin basic protein (PMBP) with dibutyryl cyclic AMP (dbcAMP) or forskolin and subsequently removed the dbcAMP or forskolin and measured the proliferative response of the clones to antigen and antigen-presenting cells (APC) in the presence or absence of exogenously added interleukin-2 (IL-2). Cells precultured with dbcAMP or forskolin for 3 days did not proliferate or produce IL-2 in response to antigen and APC, but did proliferate to antigen and APC in the presence of IL-2. Cells that had not been stimulated recently with antigen/APC or IL-2 were not affected by dbcAMP, while cells stimulated recently with antigen/APC and IL-2 were susceptible to the anergizing effect of dbcAMP. These observations support the hypothesis that elevation in intracellular cAMP in antigen-activated Th1 clones, prior to subsequent culture with antigen, induces a state of anergy.