Protein kinase A type I antagonist restores immune responses of T cells from HIV-infected patients

^a Institute of Medical Biochemistry, University of Oslo, N-0317 Oslo, Norway
^b Research Institute for Internal Medicine, University of Oslo, N-0317 Oslo, Norway
^c Section for Clinical Immunology and Infectious Diseases, Medical Department A, The National Hospital, N-0027 Oslo, Norway

	ABSTRACT

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Cyclic AMP-dependent protein kinase A (PKA) type I has been established as an acute inhibitor of T cell activation. For this reason, we investigated the possible role of PKA type I in HIV-induced T cell dysfunction. T cells from HIV-infected patients have increased levels of cAMP and are more sensitive to inhibition by cAMP analog than are normal T cells. A PKA type I-selective antagonist increases the impaired proliferation of T cells from HIV-infected patients to normal or subnormal levels (up to 2.8-fold). Follow-up of patients after initiation of highly active antiretroviral treatment revealed that a majority of patients have a persistent T cell dysfunction that is normalized by incubation of T cells with Rp-8-Br-cAMPS. These observations imply that increased activation of PKA type I may contribute to the progressive T cell dysfunction in HIV infection and that PKA type I may be a potential target for immunomodulating therapy.—Aandahl, E. M., Aukrust, P., Skålhegg, B. S., Müller, F., Frøland, S. S., Hansson, V., Taskén, K. Protein kinase A type I antagonist restores immune responses of T cells from HIV-infected patients. FASEB J. 12, 855–862 (1998)

Key Words: cAMP • PKA • AIDS • Rp-8-Br-cAMPS

	INTRODUCTION

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CYCLIC AMP COMPLETELY abolishes T cell proliferation induced through the T cell receptor/CD3 complex (TCR/CD3)² as well as early tyrosine phosphorylation after engagement of the antigen receptor (1, 2). We have previously shown that activation of cAMP-dependent protein kinase A (PKA) type I is necessary and sufficient to mediate the inhibitory effect of cAMP on T cell signaling and that PKA type I redistributes to and colocalizes with the antigen receptor during activation and capping of T cells (2, 3). This serves to establish PKA type I as an acute negative modulator of T cell antigen responses and clonal expansion. T cell dysfunction is an early event in the course of HIV infection and a major factor in the development of severe immunodeficiency. However, the molecular mechanisms by which HIV impairs T cell function have not been revealed. Two recent publications indicate that HIV-derived peptides may increase cAMP levels in vitro (4, 5). Furthermore, cAMP treatment increased HIV reverse transcriptase activity by 5- to 10-fold in a cultured T cell line (6). This may serve to establish a circulus vitiosus in the HIV-infected T cell. However, a link between the cAMP signaling pathway and the HIV-induced T cell dysfunction has not yet been established. Therefore, we investigated the possible role of cAMP-mediated inhibition of T cell immune function in purified T cells from HIV-infected patients prior to and during highly active antiretroviral therapy. We demonstrate that increased activation of PKA type I significantly inhibits T cell proliferation in cells from HIV-infected individuals independent of ongoing potent antiretroviral therapy and that this effect can be reversed by a specific antagonist of PKA type I.

	MATERIALS AND METHODS

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Patients
Nine HIV-infected patients that came to an outpatient clinic consecutively were examined, independent of clinical status, for cAMP levels in T cells ( Fig. 1A) (six men and three women: median age 40 years, range 33 to 44 years).

View larger version (12K): [in this window] [in a new window]   Figure 1. A) Levels of endogenous cAMP in peripheral blood CD3+ T cells from normal, healthy blood donors (n=10) and HIV-infected patients (n=9). Median values (horizontal lines) and single patient data (open circles) are shown. *P<0.05. B) TCR/CD3-stimulated proliferation of peripheral blood CD3+ T cells from normal, healthy blood donors and HIV-infected patients was assessed as [3H]thymidine incorporation in the presence of increasing concentrations of 8-(4-chlorophenylthio)cAMP (8-CPT-cAMP). Curve-fit analyses were performed and IC50 values were calculated (see Table 1 and Table 2 for statistical and single patient data). Normalized levels of proliferation of T cells from a healthy blood donor (open circles) and a representative HIV-infected patient (filled circles, patient #10 in Table 2) are shown (IC50 values 6.11 vs. 1.78 µM). Note left-shift of IC50 (arrow) and altered curve slope. The maximal levels of proliferation were decreased drastically in T cells from HIV-infected patients (see Table 1).

For the study portrayed in Table 1 and Table 2, blood samples were obtained from 18 HIV-infected patients (17 men and 1 woman: median age 37 years, range 27 to 54 years), clinically classified according to the revised criteria from Centers for Disease Control and Prevention (CDC) in groups of asymptomatic (CDC group A, n=8) and symptomatic (non-AIDS, CDC group B, n=4; and AIDS, CDC group C, n=6) HIV infection. Patients with ongoing acute infections or exacerbation of chronic infections at the time of blood collection were not included in the study. At the time of blood sampling, 14 patients were receiving antiretroviral therapy with didanosine (n=2), zidovudine (n=5), or a combination of zidovudine and lamivudine (n=5) or zidovudine and didanosine (n=2). None of these patients were receiving HIV protease inhibitors. Several patients received Pneumocystis carinii prophylaxis with aerosolized pentamidine (n=3), dapsone (n=7), or trimethoprim-sulfamethoxazole (n=4). Controls were eight sex- and age-matched, HIV-seronegative, healthy blood donors.

To study patients receiving highly active antiretroviral treatment ( Table 1 and Table 3) with HIV protease inhibitor indinavir (800 mg x 3 per day) in combination with zidovudine (250 mg x 2 per day) and lamivudine (150 mg x 2 per day), blood samples were obtained from nine HIV-infected patients (seven men and two women: median age 36 years, range 27 to 57 years) with symptomatic disease as classified above. Median time of treatment was 8 months (range of 4 to 12 months). T cells from four of these patients (labeled a–d) were also examined before treatment with HIV protease inhibitor, and these patients are therefore included in both Tables 2 and 3 (median time between examinations, 12 months; range, 11 to 13 months). A distinct reduction in plasma viral load was observed after initiation of therapy (median reduction in HIV RNA copies/ml: 2.44 log₁₀; range, 1.56 to 3.77 log₁₀) in all patients; at the time of blood sampling, all but three (2070, 3590, and 3800 HIV RNA copies/ml) had viral load below the detection limit of the assay. CD4⁺ lymphocyte count and viral load were determined for all patients (Table 1).

For Northern blot analyses, CD3⁺ T cells were purified separately from 10 patients with symptomatic HIV infection (seven men and three women: median age 41 years, range 35 to 49 years; several samples were collected at different time points for all patients) and pooled. T cells from five individual patients were examined by immunoblot analysis (all men: median age 42 years, range 36 to 54 years; two with asymptomatic and three with symptomatic HIV infection, as indicated in the legend to Fig. 2B).

View larger version (54K): [in this window] [in a new window]   Figure 2. Levels of protein kinase A. A) Northern blot analyses of mRNA levels of PKA subunits in normal blood donors (lane 1) and symptomatic HIV-infected patients (lane 2). Total RNA was extracted and subjected to Northern blot analyses (20 µg in each lane); resulting filters were hybridized with 32P-labeled probes to the PKA subunits RI, RII, C, and Cß. The migration of 28S and 18S rRNAs are indicated by arrowheads. Signal intensity was evaluated by densitometric scanning of suitably exposed autoradiograms and corrected for differences in loading by densitometric scanning of photographs of ethidium bromide-stained gels prior to transfer. B) Immunoblot analyses of PKA subunits. Examination of CD3+ T cells from two normal blood donors (lanes 1 and 2), two individual HIV-infected patients with asymptomatic HIV infection (lanes 3 and 4), and three patients with symptomatic HIV infection (lanes 5–7). Arrowheads indicate the migration of purified recombinant standards for PKA subunits. Equal loading was verified by densitometric scanning of Coomassie-stained gels. Densitometric analyses of immunoblots indicated either no (RI, C) or only minor (RII, 15% decrease; lanes 1 and 2 vs. lanes 5 to 7) differences in protein levels of patient samples vs. controls.

Negative selection of peripheral blood CD3⁺ T cells
Peripheral blood CD3⁺ T cells were purified by negative selection from 50 ml of heparin-treated blood from normal, healthy donors (Ullevaal University Hospital Blood Center, Oslo, Norway) or patients. Briefly, peripheral blood mononuclear cells were isolated by density gradient (Lymphoprep, NycoMed, Oslo, Norway) centrifugation, followed by negative selection using monodisperse magnetic beads directly coated with antibodies to CD14 and CD19 and rat anti-mouse immunoglobulin G (IgG) beads coated with antibodies to CD56 and a magnet. Magnetic beads were all from Dynal (Oslo, Norway; cat. no. 111.12, 111.04, and 110.11, respectively) whereas anti-CD56 antibody was from Pharmingen (San Diego, Calif.; cat. no. 31660.d). All steps were performed at 4°C. Cell suspensions were routinely screened by flow cytometry and shown to consist of more than 90% CD3⁺ and low levels of CD14⁺ (<2%), CD19⁺ (<2%), and CD56⁺ (<5%) cells.

Cyclic AMP quantitation
Levels of endogenous cAMP were examined in peripheral blood CD3⁺ T cells. CD3⁺ T cells were isolated at 4°C by negative selection; triplicate samples (2x10 cells) were harvested, followed by extraction of cAMP and analysis of intracellular cAMP content, as described elsewhere (8). Basal levels of cAMP were shown to be stable at 4°C both in crude peripheral blood mononuclear cells and CD3⁺ T cells for more than 120 min (the interval required for purification of CD3⁺ T cells; data not shown).

Proliferation assays
Proliferation assays were performed by incubating 0.075 x 10 CD3⁺ T cells/ml in a 100 µl volume in flat-bottom 96-well microtiter plates. Activation was achieved by subsequent addition of monodisperse magnetic beads coated with sheep anti-mouse IgG (Dynal, cat. no. 110.02) at a cell:bead ratio of 1:1, followed by addition of anti-CD3 (clone SpvT₃b) at a final dilution of 1:125,000 for the experiments shown. The optimal concentration of antibody was titrated carefully in the initial setup and parallel experiments at several different dilutions of antibody were always performed. Proliferation was analyzed by incubating cells for 72 h; [H]thymidine was included for the last 16 h of this period. Cells were washed and harvested onto filters with a Scatron harvester (Suffolk, U.K.) and subsequently analyzed by ß-scintillation counting. cAMP analogs, when used, were added 30 min before activation by the addition of anti-CD3 antibodies. 8-(4-Chlorophenylthio)cAMP (8-CPT-cAMP) was from Sigma (St. Louis, Mo.); Sp-8-bromo cAMP-phosphorothioate (Sp-8-Br-cAMPS) and Rp-8-Br-cAMPS were from BioLog Life Science Company (Bremen, Germany) and were dissolved to stock concentrations of 10 mM in phosphate-buffered saline (PBS); concentrations were calculated according to the extinction coefficients given by the manufacturer.

Viral load
Viral load was measured in plasma by reverse polymerase chain reaction of HIV-RNA using a commercially available kit (Amplicor HIV Monitor, Roche Diagnostic Systems, Branchburg, N.J.) and calculated as HIV-RNA copies/ml plasma (detection limit: 200 copies/ml).

Statistical analyses
For comparison of two groups of individuals, the Mann-Whitney U test (two-tailed) was used. Coefficients of correlation (R) were calculated by the Spearman's rank test. Statistical and curve fit analyses were performed using Statistica (Statsoft Inc., Tulsa, Okla.) and Sigma Plot (Jandel Corporation, Erkrath, Germany) software packages, respectively. Results are given as medians and 25th to 75th percentiles if not otherwise stated; P values are two-sided and considered significant when <0.05.

Northern blot analyses
Total RNA was extracted from CD3⁺ T cells from 10 HIV-infected patients with symptomatic HIV infection (total 75x10 cells purified separately and pooled) and from three normal blood donors (60x10 cells purified separately and pooled) by lysis in guanidium isothiocyanate and cesium-chloride gradient centrifugation, as described elsewhere (9). RNA (20 µg/lane) was separated by electrophoresis in formamide agarose gels blotted onto nylon filters and hybridized with P-labeled cDNA probes to PKA subunits, as described previously (9).

Immunoblot analyses
Purified CD3⁺ cells (2x10 cells) from five individual HIV patients and two normal blood donors were lysed directly in sodium dodecyl sulfate-polyacrylamide gel elctrophoresis (SDS-PAGE) loading buffer and subjected to SDS-PAGE and Western blotting. Resulting nitrocellulose filters were blocked in 5% bovine serum albumin with 0.05% Tween-20 in PBS and incubated in the same solution with monoclonal antibody to RI (9), anti-peptide antiserum to human RII (2), or anti-peptide antibody to C, which recognizes all human C subunits (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.; no. SC-905), all diluted 1:1000. Filters were washed overnight in PBS with 0.3% Triton X-100/0.05% Tween-20, subsequently incubated with horseradish peroxidase-labeled protein A (Amersham, Buckinghamshire, U.K.; cat. no. NA9120), and developed using enhanced chemiluminence (Amersham).

Densitometric scanning
Signal intensities of suitably exposed autoradiograms and ethidium bromide-stained gels were estimated by the use of a densitometer (Omnimedia Scanner XRS with Bioimage software, Ann Arbor, Mich.).

	RESULTS

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Elevated levels of cAMP in T cells from HIV-infected individuals
Endogenous levels of cAMP were significantly elevated in negatively selected, highly purified T cells from nine consecutive HIV-infected patients (independent of clinical status) compared to the levels in CD3⁺ T cells concomitantly isolated from ten HIV seronegative blood donors (1238 vs. 688 fmol/10 cells, P<0.05; see Fig. 1A). Furthermore, the effect of cAMP agonist on TCR/CD3-induced proliferation was investigated in 18 individual HIV-infected patients not receiving any potent antiretroviral therapy with HIV protease inhibitor and in 8 seronegative controls ( Table 1and Table 2). The patients were classified into two groups, one with asymptomatic and the other with symptomatic HIV infection (AIDS and non-AIDS) ( Table 1), according to ref 7. T cells from HIV-infected patients revealed a highly significant increase in sensitivity to inhibition of cell proliferation by exogenously added 8-CPT-cAMP ( Fig. 1B and Table 1and Table 2, P<0.001, n=17). Moreover, when the maximal proliferation rates of T cells from HIV-infected patients and that of seronegative T cells were normalized to 100% ( Fig. 1B and data not shown), it was evident that in addition to a distinct left-shifted cAMP inhibition curve, the slopes of the curves were significantly different [Hill coefficients of 1.18 (1.12–1.37), n = 17 for T cells from HIV-seropositive individuals vs. 1.59 (1.40–1.81), n = 8, for normal T cells ( Table 1; P<0.01]. The increased sensitivity to inhibition by cAMP analog suggests a contribution from elevated endogenous cAMP in priming cAMP binding site B of PKA type I, with a subsequent increase in the affinity of the A site for the exogenously added cAMP analog. The shift in curve slope from a cooperative, two-ligand site binding situation to an apparent noncooperative inhibition curve by 8-CPT-cAMP also indicates B site occupancy by endogenous cAMP.

Unchanged levels of PKA type I in CD3⁺ T cells from HIV-infected patients
Next, we examined the levels of PKA subunits in HIV-infected patients compared to normal blood donors. Figure 2A shows mRNA levels of PKA subunits in total RNA extracted from three blood donors (lane 1) and total RNA extracted from CD3⁺ cells from ten patients with symptomatic HIV infection. No changes were seen in mRNA levels of RI and Cß in HIV-infected patients compared to normal blood donors. Whereas a slight increase (20%) was seen in mRNA for RII in patients with symptomatic HIV infection, a 20% decrease was seen in C mRNA levels. Immunoblot analyses ( Fig. 2B) also demonstrated that the levels of RI protein (upper panel) were unchanged in asymptomatic patients (lanes 3 and 4) as well as in symptomatic patients (lanes 5–7) when compared to two normal blood donors (lanes 1 and 2). Protein levels for RII (middle panel) revealed very minor changes (15% decrease) in patients with symptomatic HIV infection compared to normal controls, as evaluated by densitometric scanning. C subunit levels were unchanged as detected by an antibody reactive with both C and Cß (lower panel). Thus, levels of PKA I constituted by RI and C or Cß appeared unchanged, and only very moderate changes were seen in the levels of PKA II (constituted by RII and C) in HIV-infected patients.

PKA type I antagonist improves T cell proliferation of T cells from HIV-infected patients
To further assess the specificity of the inhibition of TCR/CD3-induced T cell proliferation, we used a sulfur-substituted cAMP analog (Rp-8-Br-cAMPS) working as a full antagonist for PKA type I (10). Figure 3A shows that in T cells from normal blood donors, TCR/CD3-stimulated proliferation was inhibited by a cAMP agonist (Sp-8-Br-cAMPS). This effect was almost completely reversed by increasing concentrations of the complementary antagonist (Rp-8-Br-cAMPS). However, antagonist alone did not alter proliferation of normal T cells ( Fig. 3B). In contrast, when TCR/CD3-induced proliferation of T cells from an HIV-infected patient was investigated, we observed that not only did the antagonist (Rp-8-Br-cAMPS) reverse the effect of the complementary agonist, but further increased the proliferation above the levels in untreated cells ( Fig. 3C). When the effect of the cAMP antagonist alone was assessed in T cells from HIV-infected patients, we observed a concentration-dependent increase in TCR/CD3-induced proliferation of more than twofold at higher concentrations ( Fig. 3D). The degree of increased proliferation after treatment with cAMP antagonist was inversely correlated with the level of TCR/CD3-induced proliferation in the absence of antagonist (P<0.001, R=0.78, n=18, Table 1): T cells responding poorly to TCR/CD3 stimulation benefited most from cAMP antagonist treatment. The stimulatory effect of the cAMP antagonist was not saturated even at the highest concentrations used [ Fig. 3D; similar data (not shown) were obtained for all patients portrayed in Tables 2 and 3]. This indicates that the solubility of the compound, affinity, or availability to cells may be limiting for the effect observed. Thus, a more permeable and potent PKA type I antagonist, when available, may further improve TCR/CD3-induced proliferation of T cells from HIV-infected patients.

View larger version (65K): [in this window] [in a new window]   Figure 3. Inhibition of TCR/CD3+-stimulated T cell proliferation by the cAMP agonist Sp-8-bromo cAMP-phosphorothioate (Sp-8-Br-cAMPS) and reversal of inhibition by the complementary cAMP antagonist Rp-8-bromo-cAMP-phosphorothioate (Rp-8-Br-cAMPS) were assessed in normal, healthy blood donors (A) and HIV-infected patients (C). The effect of increasing doses of cAMP antagonist on TCR/CD3-stimulated proliferation of CD3+ T cells isolated from normal blood donors (B) and HIV-infected patients (D) was examined separately in the same experiments. A, B) Proliferation of CD3+ T cells (purified and pooled) from three healthy blood donors. C, D) Proliferation of T cells from one patient with symptomatic HIV infection (patient #1 in Table 2). Mean values of triplicate determinations ± SD are shown. See Table 1 for summarized patient data (n=18). Note: scale differs in upper vs. lower panels.

Patients on highly active antiretroviral therapy have a persistent T cell dysfunction that can be improved with PKA type I antagonist
Recently, HIV protease inhibitors have been found to slow the progression of HIV-1 disease and to strongly reduce levels of plasma HIV RNA (11, 12). For this reason, we examined T cell proliferation, cAMP sensitivity, and the effect of cAMP antagonist on T cells from nine symptomatic HIV-infected patients receiving potent antiretroviral treatment with the HIV protease inhibitor indinavir in combination with nucleoside analogs. The TCR/CD3-induced proliferative response was increased compared to untreated patients with symptomatic HIV infection (P<0.05; Table 1, lower part, and Table 3). However, the immune response of T cells from treated patients was still significantly reduced compared to normal controls (P<0.001), indicating that the HIV-specific T cell dysfunction persists in spite of potent antiretroviral treatment. Furthermore, sensitivity to inhibition by cAMP was still significantly increased compared to normal controls (P<0.01), and incubation of T cells from patients on highly active antiretroviral therapy with cAMP antagonist significantly improved TCR/CD3-induced T cell proliferation compared to that of T cells from normal individuals (P<0.05). Single patient data from this group revealed heterogeneity among the patients receiving potent antiretroviral therapy ( Table 3). Proliferation of T cells from six of nine patients benefited from Rp-8-Br-cAMPS in a dose-dependent manner (1.5- to 2.8-fold increase in immune response) whereas T cells from three patients with subnormal proliferative response did not respond to cAMP antagonist (proliferation 0.98- to 1.11-fold of that in untreated cells). This is reflected in the inverse correlation of the level of TCR/CD3-induced proliferation in the absence of antagonist and the effect of treatment with the cAMP antagonist (P<0.01, R=0.81, n=9): only T cells from patients with persistent T cell dysfunction benefited from treatment with cAMP antagonist. A follow-up of 4 of the 18 patients examined in Table 1and Table 2after initiation of highly active antiretroviral therapy showed increased proliferative response of T cells after onset of treatment (compare Tables 2 and 3). However, T cells from two of the patients remained severely suppressed in immune response (labeled a and b), and T cell proliferation from these patients still benefited substantially from incubation with cAMP antagonist. Cells from the two other patients (labeled c and d) reached subnormal levels of T cell proliferation after the onset of potent antiretroviral therapy and did not benefit further from incubation with Rp-8-Br-cAMPS.

	DISCUSSION

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The results described here demonstrate elevated levels of cAMP in T cells from untreated HIV-infected patients. Furthermore, use of a cAMP antagonist preferentially acting on PKA type I greatly improves the immune response of T cells from these patients, as assessed by an increase in proliferative response. Although treatment of peripheral blood mononuclear cells from HIV-infected individuals with interleukin 12 (IL-12) or anti-IL-10 has been shown to improve immune responses (13, 14), the finding that treatment of T cells from HIV-infected patients with cAMP/PKA type I antagonist restores immune responses is the first demonstration of a potential target for immunomodulation in the T cell itself. Levels of PKA type I were unaltered in T cells from HIV-infected patients compared to normal controls according to immunoblot analysis. With total levels of PKA type I apparently unchanged and increased basal levels of endogenous cAMP, PKA type I will be constitutively activated. However, alterations in cAMP levels and PKA subunits may be confined to distinct subsets of CD3⁺ T cells.

Previous observations demonstrate that PKA type I not only colocalizes with antigen receptor in T cells, but localizes similarly in B cells and completely abolishes mitogenic responses in both T and B cells and the cytotoxic function of natural killer (NK) cells (2, 3, 15–18). Together with the observations that triggering of the TCR/CD3 complex leads to production of cAMP (19, 20), this prompted us to hypothesize that the normal lymphocyte immune responsiveness is negatively modulated by cAMP through PKA type I and that PKA activation after antigen receptor triggering is a negative feedback mechanism. This is further supported by the recent observations that PKA type I is impaired in T cells from patients with systemic lupus erythematosus, suggesting that the lack of cAMP/PKA type I-mediated immunomodulation may lead to an overshoot of immune cell responses, thus contributing to the pathogenesis of this autoimmune disease (21, 22). It appears that elevated levels of cAMP in T cells from HIV-infected patients shift the equilibrium in the opposite direction and produce a situation where constant inhibition through PKA type I significantly impairs the immune responsiveness of T cells in vitro. This is further supported by the recent observation that cAMP is elevated in crude peripheral blood mononuclear cells from HIV-infected patients containing a mixture of B, T, and NK cells as well as monocytes (23).

Future studies addressing mechanisms that elevate cAMP in T cells from HIV-infected patients will be of great interest and may help us understand the pathogenetic impact of PKA type I dysregulation on HIV-induced immunodeficiency in vivo. Although highly active antiretroviral therapy is now available, the failure of these treatment regimens to totally eradicate the virus (24), the emergence of drug-resistant strains (25), the rapid relapse upon withdrawal of therapy (24), and the persistent HIV-specific immunodeficiency as demonstrated here call for additional compensatory immunomodulating therapy. Despite markedly reduced or undetectable levels of HIV RNA in patients on highly active antiretroviral therapy, a majority of the patients had a persistent T cell dysfunction that could be reversed by incubation with a PKA type I-selective antagonist. This demonstrates a potential target for immunomodulation. Treatment regimens that counteract the activation of PKA type I may be a supplement to potent antiretroviral therapy for HIV-infected patients with persistently impaired T cell function.

	ACKNOWLEDGMENTS

 
This work was supported by the Norwegian Cancer Society, the Norwegian Research Council, Anders Jahres Foundation for the Promotion of Science, the Novo Nordisk Research Committee, the Pasteur Legacy, and Odd Kåre Rabben's Memorial Fund for AIDS research.

	FOOTNOTES

 
¹ Correspondence: Institute of Medical Biochemistry, University of Oslo, P.O. Box 1112, Blindern, N-0317 Oslo, Norway. E-mail: kjetil.tasken{at}basalmed.uio.no

² Abbreviations: Sp-8-Br-cAMPS, Sp-8-bromo cAMP-phosphorothioate; PBS, phosphate-buffered saline; SD-PAGE, sodium dodecyl sulfate-polyacrylamide gele elctrophoresis; IL, interleukin; NK, natural killer; PKA, protein kinase A; CDC, Centers for Disease Control and Prevention; TCR/CD3, T cell receptor/CD3 complex; IgG, immunoglobulin G; 8-CPT-cAMP, 8-(4-chlorophenylthio)cAMP.

Received for publication November 21, 1997. Accepted for publication January 26, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Kammer, G. M. (1988) The adenylate cyclase-cAMP-protein kinase A pathway and regulation of the immune response. Immunol. Today 9, 222–229[Medline]
2. Skålhegg, B. S., Landmark, B. F., Døskeland, S. O., Hansson, V., Lea, T., and Jahnsen, T. (1992) Cyclic AMP-dependent protein kinase type I mediates the inhibitory effects of 3',5'-cyclic adenosine monophosphate on cell replication in human T lymphocytes. J. Biol. Chem. 267, 15707–15714[Abstract/Free Full Text]
3. Skålhegg, B. S., Taskén, K., Hansson, V., Huitfeldt, H. S., Jahnsen, T., and Lea, T. (1994) Location of cAMP-dependent protein kinase type I with the TCR/CD3 complex. Science 263, 84–87[Abstract/Free Full Text]
4. Hofmann, B., Nishanian, P., Nguyen, T., Insixiengmay, P., and Fahey, J. L. (1993) Human immunodeficiency virus proteins induce the inhibitory cAMP/protein kinase A pathway in normal lymphocytes. Proc. Natl. Acad. Sci. USA 90, 6676–6680[Abstract/Free Full Text]
5. Haraguchi, S., Good, R. A., and Day, N. K. (1995) Immunosuppressive retroviral peptides: cAMP and cytokine patterns. Immunol. Today 16, 595–603[Medline]
6. Nokta, M. A., and Pollard, R. B. (1992) Human immunodeficiency virus replication: modulation by cellular levels of cAMP. AIDS Res. Hum. Retroviruses 8, 1255–1261[Medline]
7. Center for Disease Control and Prevention. (1992) 1993: Revised classification for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. Morbid. Mortal. Wkly. Rep. 41, 1
8. Taskén, K., Andersson, K. B., Skålhegg, B. S., Taskén, K. A., Hansson, V., Jahnsen, T., and Blomhoff, H. K. (1993) Reciprocal regulation of mRNA and protein for subunits of cAMP-dependent protein kinase (RI and C) by cAMP in a neoplastic B cell line (Reh). J. Biol. Chem. 268, 23483–23489[Abstract/Free Full Text]
9. Taskén, K., Skålhegg, B. S., Solberg, R., Andersson, K. B., Taylor, S. S., Lea, T., Blomhoff, H. K., Jahnsen, T., and Hansson, V. (1993) Novel isozymes of cAMP-dependent protein kinase exist in human cells due to formation RI-RIß heterodimeric complexes. J. Biol. Chem. 268, 21276–21283[Abstract/Free Full Text]
10. Gjertsen, B. T., Mellgren, G., Otten, A., Maronde, E., Genieser, H. G., Jastorff, B., Vintermyr, O. K., McKnight, G. S., and Døskeland, S. O. (1995) Novel (Rp)-cAMPS analogs as tools for inhibition of cAMP-kinase in cell culture. J. Biol. Chem. 270, 20599–20607[Abstract/Free Full Text]
11. Hammer, S. M., Squires, K. E., Hughes, M. D., Grimes, J. M., Demeter, L. M., Currier, J. S., Eron, J. J. J., Feinberg, J. E., Balfour, H. H. J., Deyton, L. R., Chodakewitz, J. A., and Fischl, M. A. (1997) A controlled trial of two nucleoside analogues plus indinavir in persons with human immunodeficiency virus infection and CD4 cell counts of 200 per cubic millimeter or less. N. Engl. J. Med. 337, 725–733[Abstract/Free Full Text]
12. Gulick, R. M., Mellors, J. W., Havlir, D., Eron, J. J., Gonzalez, C., McMahon, D., Richman, D. D., Valentine, F. T., Jonas, L., Meibohm, A., Emini, E. A., and Chodakewitz, J. A. (1997) Treatment with indinavir, zidovudine, and lamivudine in adults with human immunodeficiency virus infection and prior antiretroviral therapy. N. Engl. J. Med. 337, 734–739[Abstract/Free Full Text]
13. Clerici, M., Lucey, D. R., Berzofsky, J. A., Pinto, L. A., Wynn, T. A., Blatt, S. P., Dolan, M. J., Hendrix, C. W., Wolf, S. F., and Shearer, G. M. (1993) Restoration of HIV-specific cell-mediated immune responses by interleukin-12 in vitro. Science 262, 1721–1724[Abstract/Free Full Text]
14. Landay, A. L., Clerici, M., Hashemi, F., Kessler, H., Berzofsky, J. A., and Shearer, G. M. (1996) In vitro restoration of T cell immune function in human immunodeficiency virus-positive persons: effects of interleukin (IL)-12 and anti-IL-10. J.Infect. Dis. 173, 1085–1091[Medline]
15. Laxminarayana, D., Berrada, A., and Kammer, G. M. (1993) Early events of human T lymphocyte activation are associated with type I protein kinase A activity. J. Clin. Invest. 92, 2207–2214
16. Laxminarayana, D., and Kammer, G. M. (1996) Activation of type I protein kinase A during receptor-mediated human T lymphocyte activation. J. Immunol. 156, 497–506[Abstract]
17. Levy, F. O., Rasmussen, A. M., Taskén, K., Skålhegg, B. S., Huitfeldt, H. S., Funderud, S., Smeland, E. B., and Hansson, V. (1996) Cyclic AMP-dependent protein kinase (cAK) in human B cells: co-localization of type I cAK (RI₂C₂) with the antigen receptor during anti-immunoglobulin-induced B cell activation. Eur. J. Immunol. 26, 1290–1296[Medline]
18. Torgersen, K. M., Vaage, J. T., Levy, F. O., Hansson, V., Rolstad, B., and Taskén, K. (1997) Selective activation of cAMP-dependent protein kinase type I inhibits rat natural killer cell cytotoxicity. J. Biol. Chem. 272, 5495–5500[Abstract/Free Full Text]
19. Ledbetter, J. A., Parsons, M., Martin, P. J., Hansen, J. A., Rabinovitch, P. S., and June, C. H. (1986) Antibody binding to CD5 (Tp67) and Tp44 T cell surface molecules: effects on cyclic nucleotides, cytoplasmic free calcium, and cAMP-mediated suppression. J. Immunol. 137, 3299–3305[Abstract]
20. Kammer, G. M., Boehm, C. A., Rudolph, S. A., and Schultz, L. A. (1988) Mobility of the human T lymphocyte surface molecules CD3, CD4, and CD8: regulation by a cAMP-dependent pathway. Proc. Natl. Acad. Sci. USA 85, 792–796[Abstract/Free Full Text]
21. Kammer, G. M., Khan, I. U., and Malemud, C. J. (1994) Deficient type I protein kinase A isozyme activity in systemic lupus erythematosus T lymphocytes. J. Clin. Invest. 94, 422–430
22. Dayal, A. K., and Kammer, G. M. (1996) The T cell enigma in lupus [Review]. Arthritis Rheum. 39, 23–33[Medline]
23. Hofmann, B., Nishanian, P., Nguyen, T., Liu, M., and Fahey, J. L. (1993) Restoration of T-cell function in HIV infection by reduction of intracellular cAMP levels with adenosine analogues. AIDS 7, 659–664[Medline]
24. Pantaleo, G. (1997) How immune-based interventions can change HIV therapy. Nat. Med. 3, 483–486[Medline]
25. Condra, J. H., Schleif, W. A., Blahy, O. M., Gabryelski, L. J., Graham, D. J., Quintero, J. C., Rhodes, A., Robbins, H. L., Roth, E., and Shivaprakash, M. (1995) In vivo emergence of HIV-1 variants resistant to multiple protease inhibitors. Nature (London) 374, 569–571[Medline]