Decreased Dipeptidyl Peptidase IV Enzyme Activity of Plasma Soluble CD26 and Its Inverse Correlation with HIV-1 RNA in HIV-1 Infected Individuals

Clinical Immunology Vol. 91, No. 3, June, pp. 283–295, 1999 Article ID clim.1999.4711, available online at http://www.idealibrary.com on

Decreased Dipeptidyl Peptidase IV Enzyme Activity of Plasma Soluble CD26 and Its Inverse Correlation with HIV-1 RNA in HIV-1 Infected Individuals Osamu Hosono,* Toshio Homma,* Hiroshi Kobayashi,* Yasuhiko Munakata,† Yoshihisa Nojima,* Aikichi Iwamoto,‡ and Chikao Morimoto* ,† ,1 *Department of Clinical Immunology and AIDS Research Center and ‡Department of Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo 108-0071, Japan; and †Division of Tumor Immunology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115

Human plasma contains soluble CD26/dipeptidyl peptidase IV (sCD26/DPPIV) although its physiological significance remains unclear. To determine whether the plasma sCD26 levels have clinical relevance in HIV-1 infected individuals, the concentration and DPPIV enzyme activity of plasma sCD26 were measured. While there is no significant difference between the plasma levels of sCD26 in 90 HIV-1 infected individuals and in 79 uninfected controls, specific DPPIV enzyme activity of sCD26 was significantly decreased in HIV-1 infected individuals (P < 0.0001). Specific DPPIV enzyme activity was correlated with the levels of CD4 1 T cells (r 5 0.247; P < 0.02), CD8 1 T cells (r 5 0.236; P < 0.03), and adenosine deaminase (r 5 0.227; P < 0.05) and had an inverse correlation with HIV-1 RNA (Spearman’s r 5 0.474; P 5 0.0012). Furthermore, recombinant sCD26 enhanced the in vitro PPD-induced response of lymphocytes from HIV-1 infected individuals with decreased specific DPPIV enzyme activity. These results suggest that the specific DPPIV enzyme activity of plasma sCD26 may contribute to the immunopathogenesis of HIV infection. © 1999 Academic Press Key Words: soluble CD26; dipeptidyl peptidase IV; HIV-RNA. INTRODUCTION

CD26 is a widely distributed 110-kDa cell surface glycoprotein with known dipeptidyl peptidase IV (DPPIV; EC 3.4.14.5) activity in its extracellular domain (1– 4). This ectoenzyme is capable of cleaving aminoterminal dipeptides from polypeptides with either L-proline or L-alanine in position 2 (5). On T cells, 1 To whom correspondence and reprint requests should be addressed at Department of Clinical Immunology and AIDS Research Center, The Institute of Medical Science, The University of Tokyo. 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-0071, Japan. Fax: 1813-5449-5448. E-mail: [email protected].

CD26 expression is preferentially restricted to the CD4 1 helper/memory population, and CD26 can deliver a potent T cell costimulatory activation signal. Crosslinking of CD26 and CD3 with solid-phase immobilized mAbs can induce T cell costimulation and interleukin 2 production by either human CD4 1 T cells or Jurkat T cell lines transfected with CD26 cDNA (6, 7). Moreover, we have demonstrated that DPPIV enzyme activity is required for CD26-mediated T cell costimulation (7). The cDNA sequence of CD26 predicts a type II membrane protein with only six amino acids in its cytoplasmic region, suggesting that in addition to DPPIV enzyme activity, other signal-inducing molecules may be associated with CD26 (8). There is evidence that CD26 interacts, presumably through its extracellular domain, with both CD45, a protein tyrosine phosphatase, and adenosine deaminase (ADA), each of which is capable of functioning in a signal transduction pathway (9, 10). To further determine how CD26 functions in T cell costimulation, we have prepared recombinant soluble molecules comprising only the extracellular domain of CD26 and have shown that recombinant soluble CD26 (rsCD26) enhanced proliferative responses of PBLs to stimulation with soluble antigen (11). This enhancing effect required DPPIV enzyme activity. However, this enhancing effect on T cell responses to the recall antigen by rsCD26 was not observed in all individuals. Individuals with high plasma soluble CD26 (sCD26) and with a pronounced response to tetanus toxoid were insensitive to the effects of added rsCD26 (11). This observation supports the view that plasma sCD26 may have already modulated the T cell response of individuals in vivo and suggests that sCD26 has biologically important immunomodulatory activity and is relevant in immunodeficiency disorders. CD4 1 lymphocytes in patients with AIDS have an intrinsic defect in their ability to recognize and respond

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to “recall antigens” sometime before a reduction in the total number of CD4 1 cells occurs (12, 13). The response to recall antigens is clearly a property of CD4 1CD26 1 T cells, since this is the only helper population known to proliferate in response to soluble antigens and to induce both MHC-restricted cytotoxic T lymphocytes capable of killing virus-infected target cells and B cells to secrete immunoglobulins (2). Each of these properties is a key to the host’s response to viral infection. In this regard, a selective decrease in CD26 1 T cells has been reported in HIV-1 infected individuals prior to a general decrease in CD4 1 T cells (14, 15). Moreover, Tat, a regulatory protein encoded by the HIV-1 genome (16), has been shown to suppress the response of human peripheral T cells to soluble antigens (17). It has also been suggested that Tat can bind to CD26 and partially inhibit DPPIV enzyme activity (18). Hovanessian and colleagues (19) have proposed that CD26 and DPPIV enzyme activity serve as essential cofactors for HIV-1 entry, but our previous studies (20) as well as others (21, 22) have shown that CD26 is not a necessary cofactor for HIV-1 infection. Although it has been shown that chemokine receptors are co-receptors for HIV infection (23–25), CD26/ DPPIV still appears to regulate HIV entry and apoptosis (20, 26). Since sCD26 is present in the plasma and appears to have important immunoregulatory effects, we attempted to determine whether the plasma sCD26 levels have clinical relevance in HIV-1 infected individuals. Here we show that DPPIV enzyme activity of plasma sCD26 was decreased in HIV-1 infected individuals and inversely correlated with HIV-1 RNA, and that the in vitro addition of rsCD26 could enhance purified protein derivative (PPD)-induced lymphocyte proliferation, especially in those with a CD4 1 T cell count greater than 100/mm 3, a weak response to PPD, and a decreased specific DPPIV enzyme activity of plasma sCD26. These results suggest that specific DPPIV enzyme activity of plasma sCD26 in HIV-1 infected individuals contributes to the immunopathogenesis of HIV infection. METHODS

Subjects Studied HIV-1 infected individuals investigated in this study were selected from those referred to the Research Hospital, Institute of Medical Science, University of Tokyo, Tokyo, Japan. Ninety HIV-1 infected individuals (79 men and 11 women; mean age 33 years) were studied. Clinical category was according to the 1993 revised Centers for Disease Control and Prevention (CDC) classification (27). Plasma from 79 HIV-1 uninfected healthy individuals (74 men and 5 women; mean age

31 years) served as controls. All control plasma were also negative for antibodies against hepatitis virus type B and type C. The characteristics of the HIV-1 infected individuals at the time of the study are detailed in Table 1. Determination of sCD26 and DPPIV Enzyme Activity in Plasma sCD26/DPPIV detection was performed on plasma samples kept frozen at 280°C. To measure sCD26 or its DPPIV enzyme activity, Maxisorp ELISA immunoplates (Nunc A/S, Roskilde, Denmark) were coated with 100 ml of 10 mg/ml anti-CD26 mAb (5F8) in 0.05 M carbonate/bicarbonate buffer, pH 9.6, by incubating overnight at 4°C. Remaining binding sites were blocked with 200 ml of 25% Block Ace (DaiNihon Pharmaceutical Co. Ltd., Osaka, Japan) at 4°C overnight. Plates were then incubated with 100 ml of plasma appropriately diluted with 0.01 M PBS containing 0.05% Tween 20 for 2 h at room temperature. Bound sCD26 were detected by incubating with 100 ml of biotin-conjugated anti-CD26 mAb (1F7) followed by ExtrAvidin-alkaline phosphatase (Sigma Chemical Co., St. Louis, MO). The plates were developed with 1 mg/ml p-nitrophenyl phosphate in 10 mM diethanolamine buffer, pH 9.6, containing 0.5 mM MgCl 2. The color development was monitored at 405 nm on an ELISA reader (model 3550; Bio-Rad Laboratories, Hercules, CA). This immunoassay was standardized by using rsCD26 produced by transfected Chinese hamster ovary cells as described previously (11). In parallel with measuring sCD26 protein concentration, DPPIV enzyme activity of bound sCD26 was detected by incubating with the substrate, glycylproline p-nitroanilide (Gly-Pro-pNA, 1 mg/ml in PBS) (Sigma Chemical Co.), and the color development was measured at 405 nm. This assay was standardized by using a standard curve for p-nitroaniline (Sigma Chemical Co.). Results were expressed as specific DPPIV enzyme activity (nmol of p-nitroaniline formed/min/mg sCD26 protein at 25°C). Measurement of Lymphocyte Subsets White blood cell samples drawn in tubes containing heparin were stained with monoclonal antibodies and the percentages of CD4 1 and CD8 1 T cells in PBLs were determined by flow-cytometric procedures. The numbers of CD4 1 and CD8 1 T cells were determined by obtaining total and differential white blood cell counts and multiplying by the appropriate factor obtained with flow cytometry.

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TABLE 1 Characteristics of HIV-1 Infected Individuals and Uninfected Controls Transmission category

Subject group

n

Sex (M/F)

Age (years); mean (range)

Homosexual/ bisexual man

Heterosexuals

Transfusion recipients

HIV-1 infected individuals Clinical category a A1 A2 A3 B1 B2 B3 C1 C2 C3 Uninfected controls

90

79/11

33 (15–67)

29

19

42

3 35 21 0 6 5 0 2 18 79

2/1 27/8 21/0

29 (24–37) 31 (15–50) 32 (17–48)

10 6

10 4

3 15 11

4/2 5/0

31 (19–37) 48 (31–67)

3 2

2

2/0 18/0 74/5

40 (31–49) 33 (18–49) 31 (18–60)

8

3

a

1 3 2 7

Clinical category is according to the 1993 revised CDC classification.

Adenosine Deaminase Assay Plasma ADA, which catalyzes the deamination of adenosine and deoxyadenosine to inosine and deoxinosine, respectively, was measured by an autoanalyzer using the appropriate reagent (AD Auto Maruho; Maruho Co. Ltd., Osaka, Japan) according to the manufacturer’s specifications. Quantitation of HIV-1 RNA in Plasma Plasma HIV-1 RNA levels were determined by use of the quantitative Quantiplex HIV-RNA branched DNA assay (Chiron Corp., Emeryville, CA) according to the manufacturer’s specifications. Cutoff value of this assay was 10 4 HIV-1 RNA equivalents/ml. In addition, we also used the Amplicor HIV Monitor kit (Roche Molecular Systems, Branchburg, NJ) in the follow-up of some patients. The lower limit of detection for this assay is 400 copies/ml. Lymphocyte Proliferation Assays PBLs from HIV-1 infected individuals were separated from heparinized venous blood by Ficoll–Paque (Pharmacia Biotech AB, Uppsala, Sweden) density gradient centrifugation. PBLs were suspended in complete medium containing RPMI 1640 (Gibco BRL, Gaithersburg, MD) supplemented with 10% FCS, 4 mM L-glutamine, 25 mM Hepes buffer, 0.5% sodium bicarbonate, and 1% penicillin–streptomycin (Gibco BRL). Aliquots of 1 3 10 5 PBLs were cultured with PPD concentrations (0, 1, and 10 mg/ml) with or without rsCD26 (1 mg/ml) for 6 days in 96-well round-

bottom culture plates (Corning Costar Corp., Cambridge, MA). Cultured PBLs were pulsed with 1 mCi of [ 3H]thymidine overnight before harvesting. Radioactivity incorporated into DNA was measured by a direct beta counter (Matrx96; Packard Instrument Co., Meriden, CT). The PPD-specific response was determined as follows: PPD-specific response 5 (PPD cpm 2 cells alone cpm). The PPD 1 rsCD26-specific response was determined as follows: PPD 1 rsCD26-specific response 5 [(PPD 1 rsCD26 cpm) 2 rsCD26 cpm]. The enhancement index seen in the presence of rsCD26 was calculated as follows: enhancement index 5 (PPD 1 rsCD26-specific response)/(PPD-specific response). Statistical Analysis The means were calculated 6SD. Statistical comparisons between the groups were performed using the Student t test and Mann–Whitney U test, as appropriate. Correlation coefficients were calculated by Pearson’s test, except for Spearman’s rank-correlation coefficient between specific DPPIV enzyme activity and HIV-1 RNA, whose values were log 10-transformed. RESULTS

Study Population The mean age (33 years) and age range (15– 67 years) of HIV-1 infected individuals were similar to those of uninfected controls as shown in Table 1. The groups with B1 and C1 CDC clinical categories were not observed in this study. Most of the subjects were categorized into A2, A3, and C3. According to the recognized transmission categories, 29 HIV-1-positive

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subjects were homosexual/bisexual, 19 heterosexual, and 42 transfusion recipients. These recognized transmission groups showed a similar distribution of CDC clinical categories. There was no known difference between male and female patients, although the small number of female patients makes statistical analysis difficult. sCD26 Levels and Specific DPPIV Enzyme Activity in Plasma To determine plasma sCD26 levels, we examined 90 HIV-1 infected individuals and 79 uninfected controls. As shown in Fig. 1A, plasma sCD26 in HIV-1 infected individuals revealed a wider range of levels (5.9 –53.5 mg/ml) than in uninfected controls (9.5–22.1 mg/ml). However, there was no significant difference in the levels of sCD26 between HIV-1 infected individuals and uninfected controls (15.6 6 7.0 vs 14.9 6 3.1 mg/ ml). It should be noted that plasma sCD26 levels are compatible with the serum sCD26 levels. It has been shown that DPPIV enzyme activity plays an important role in costimulatory activity of CD26 in T cell activation (7). Since it is reported that HIV-1 infected individuals have defective immune function (12), it is conceivable that specific DPPIV enzyme activity may be altered in the plasma of HIV-1 infected individuals. Therefore, we simultaneously determined specific DPPIV enzyme activity of plasma sCD26. As shown in Fig. 1B, specific DPPIV enzyme activity was significantly decreased in HIV-1 infected individuals compared to that in uninfected controls (0.82 6 0.14 vs 0.95 6 0.13 nmol/min/mg sCD26, P , 0.0001). In contrast to sCD26 levels, specific DPPIV enzyme activity in HIV-1 infected individuals showed a similar distribution to that of uninfected controls. In addition, total DPPIV enzyme activity of plasma was also significantly decreased in HIV-1 infected individuals compared to uninfected controls (1.22 6 0.39 vs 1.39 6 0.22 nmol/min, P , 0.001). sCD26/DPPIV Levels and Clinical Implications To determine the clinical significance of decreased specific DPPIV enzyme activity in HIV-1 infected individuals, we examined the relationship between specific DPPIV enzyme activity and CDC clinical categories. Specific DPPIV enzyme activity was 0.85 6 0.11 nmol/min/mg sCD26 in 38 asymptomatic subjects (A1, A2), 0.81 6 0.23 nmol/min/mg sCD26 in 6 symptomatic subjects (B1, B2), and 0.80 6 0.15 nmol/min/mg sCD26 in 46 subjects with AIDS (A3, B3, C). Interestingly, it was significantly lower in subjects with AIDS-indicator conditions (C; 0.78 6

0.14 nmol/min/mg sCD26) than in asymptomatic subjects (P , 0.05). Moreover, decreased specific DPPIV enzyme activity did not correlate with the infectious states of hepatitis virus type C, hepatitis virus type B, and other complications (data not shown). Since specific DPPIV enzyme activity seemed to be decreased with the progression of disease, the correlation between specific DPPIV enzyme activity and T cell subset was next examined. As shown in Fig. 2, specific DPPIV enzyme activity shows positive correlations with levels of both CD4 1 T cells (r 5 0.247, P , 0.02, n 5 90) (Fig. 2A) and CD8 1 T cells (r 5 0.236, P , 0.03, n 5 90) (Fig. 2B). Total lymphocyte count also correlated with DPPIV enzyme activity (r 5 0.260, P , 0.02, n 5 90). These results indicate that the higher specific DPPIV enzyme activity appears to correlate with the higher CD4 and CD8 counts. We have previously shown that CD26 is an ADA binding protein (10), and ADA could regulate the local adenosine concentration which influences inflammation and immune response (28). We next investigated correlation between plasma ADA level and specific DPPIV enzyme activity. As shown in Fig. 3, the plasma ADA level had a negative correlation with specific DPPIV enzyme activity (r 5 20.227, P , 0.05, n 5 81). Moreover, the elevated levels of ADA in most HIV-1 infected individuals were observed. Since ADA is enriched in the intracellular space of T lymphocytes, the above observation suggests that the destruction of T lymphocytes appeared to be markedly increased in HIV-1 infected individuals. It should be noted that ADA does not inhibit DPPIV enzyme activity, indicating that decreased specific DPPIV enzyme activity was not induced by the direct binding of ADA to the catalytic site of sCD26. Correlation between Plasma Specific DPPIV Enzyme Activity and HIV-1 RNA Recently, it has been shown that the plasma viral load was the single best indicator of progression in HIV-1 infection as well as for efficacy of anti-HIV therapy, even more predictive than that of the CD4 1 T cell counts (29). Therefore, we quantitated plasma HIV-1 RNA in 48 HIV-1 infected individuals to compare with specific DPPIV enzyme activity in the same sample. As shown in Fig. 4, there was a significant negative correlation between specific DPPIV enzyme activity and plasma HIV-1 RNA (Spearman’s r 5 20.474, P 5 0.0012, n 5 48). This suggests that the higher specific DPPIV enzyme activity appears to correlates with the lower HIV-1 RNA levels. These results suggest that levels of specific DPPIV enzyme activity may reflect the pathologic process in HIV-1 infection and be a predictor of HIV-1 disease progression as HIV-1 RNA. To

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FIG. 1. Plasma sCD26 levels in HIV-1 infected individuals (A). Plasma sCD26 concentrations were measured by a sandwich ELISA method using two anti-CD26 mAbs (5F8, 1F7) as described under Methods. There was no significant difference between levels in HIV-1 infected individuals and in uninfected controls. Decreased specific DPPIV enzyme activity of plasma sCD26 in HIV-1 infected individuals (B). Total DPPIV enzyme activity of plasma was determined by capture on anti-CD26 mAb (5F8) coated plates followed by incubation with Gly-Pro pNA and measurement at OD 405 nm. Results were expressed as specific DPPIV enzyme activity of plasma sCD26 (nmoles of p-NA formed/min/mg sCD26).

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FIG. 2. Correlation between specific DPPIV enzyme activity and T cell subsets in peripheral blood. Specific DPPIV enzyme activity significantly correlated with the number of CD4 1 T cells (A) and CD8 1 T cells (B). The percentages of CD4 1 and CD8 1 T cells in PBLs were determined by flow-cytometric analysis. The numbers of CD4 1 and CD8 1 T cells were determined by obtaining total and differential white blood cell counts and multiplying by the appropriate factor obtained with flow cytometry.

further clarify the clinical significance of sCD26/ DPPIV, longitudinal studies were performed. Levels of specific DPPIV enzyme activity changed little during stable levels of CD4 count and HIV-1 RNA (data not shown). However, as shown in Fig. 5, effective antiHIV therapy induced not only HIV-1 RNA suppression

and the increase of CD4 count but also concomitant increase of specific DPPIV enzyme activity. These findings strongly support the notion that specific DPPIV enzyme activity of plasma sCD26 has a inverse correlation with HIV-1 RNA levels and is a potential surrogate marker.

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FIG. 3. Inverse correlation between specific DPPIV enzyme activity and plasma ADA. Plasma ADA was measured by an autoanalyzer using the appropriate reagent. In particular, plasma levels of ADA were increased in most HIV-1 infected individuals. The hatched area indicates a normal range of plasma ADA (6.8 to 18.2 U/L).

Correlation of Plasma sCD26/DPPIV Levels with the PPD-Induced Lymphocyte Response to Added rsCD26 We have shown that rsCD26 could amplify the in vitro response to recall antigens of normal individuals with low serum sCD26, and this enhancing effect required DPPIV enzyme activity (11). Therefore, we attempted to determine whether rsCD26 has an enhancing effect on T cell memory response, and if so, whether it is correlated with plasma sCD26/DPPIV levels in HIV-1 infected individuals. As shown in Table 2, the addition of rsCD26 could enhance the in vitro T cell response to PPD from HIV-1 infected individuals in Group 2 with decreased specific DPPIV enzyme activity (P , 0.04, compared with that of uninfected controls) who showed $100 CD4 1 T cells/mm 3 and ,1000 cpm of PPD response. HIV-1 infected individuals in Group 3 with $100 CD4 1 T cells/mm 3 and $1000 cpm of PPD response comparable to uninfected controls (data not shown) showed higher plasma specific DPPIV enzyme activity, thus demonstrating a minimal enhancing effect of rsCD26 on PPD response. HIV-1 infected individuals in Group 1 showed #100 CD4 1 T cells/mm 3 and minimal response to PPD stimulation, which might be due to a lack of a PPD-responsive CD4 1 T cell population. rsCD26 could enhance the PPD-induced response of lymphocytes from HIV-1 infected individuals with decreased specific DPPIV enzyme ac-

tivity, suggesting that the addition of rsCD26 might be beneficial for restoring the memory T cell response in HIV-1 infected individuals. DISCUSSION

CD26 is a multifunctional molecule involved in regulation of many key aspects of lymphocyte function. We have previously demonstrated that its DPPIV enzyme activity plays an important role in T cell costimulation (7) and relative resistance to HIV-1 infection (20). The present study demonstrates that specific DPPIV enzyme activity of plasma sCD26 was decreased in HIV-1 infected individuals and inversely correlated with HIV-1 RNA, which is reported to be the single best parameter for the prognosis of HIV infection (29). Moreover, we showed that the in vitro addition of rsCD26 could enhance PPD-induced lymphocyte proliferation, especially in those with a CD4 1 T cell count greater than 100/mm 3 and a relatively weak in vitro response to PPD. CD26 has been shown to be a binding protein for ADA (10), collagen (30), and the HIV-1 Tat protein (18). Although it is reported that collagen and ADA did not inhibit DPPIV enzyme activity, the HIV-1 Tat protein has been shown to partially inhibit DPPIV enzyme activity (18). However, subsequent studies showed that specific binding of HIV-1 Tat to CD26 under physiological condition was not observed (31). Specific DPPIV

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FIG. 4. Inverse correlation between specific DPPIV enzyme activity and HIV-1 RNA in plasma. Plasma HIV-1 RNA levels were determined using the quantitative Quantiplex HIV-RNA branched DNA assay (Chiron Corp.). The lower limit of detection of this assay is 10 4 HIV-1 RNA equivalents/ml and measurements below this are assigned a value of 10 4 HIV-1 RNA equivalents/ml.

enzyme activity of plasma sCD26 was decreased in HIV-1 infected individuals, especially in patients with AIDS. Furthermore, individuals with lower specific DPPIV enzyme activity have higher plasma HIV-1 RNA levels (Fig. 4) and our longitudinal studies show the significant increase in specific DPPIV enzyme activity together with elevation of CD4 count and decline of HIV-1 RNA after antiretroviral therapy (Fig. 5). HIV-1 related proteins including HIV-1 Tat and gp120 could influence T cell function and survival (32). There are some possible explanations for decreased specific DPPIV enzyme activity in HIV-1 infected individuals. One possibility is that HIV-1 infection may induce the alteration of the antigen epitope in CD26 and therefore specific binding of HIV-1 Tat to CD26 under physiological conditions may occur, resulting in the reduced specific DPPIV enzyme activity. Alternatively, other unknown HIV-1 related CD26 binding proteins in the sera may interfere with the enzymatic activity of CD26/DPPIV. Elucidation of the precise mechanism of decreased DPPIV activity in such patients’ plasma should further contribute to a better understanding of the immunopathogenesis in HIV infection. Plasma sCD26 in HIV-1 infected individuals showed a wide range of levels. In some subjects, a significant increase in sCD26 levels with low specific DPPIV enzyme activity was observed during disease progression.

It was previously reported that the DPPIV activity in lysates from total PBL was shown to be significantly reduced in HIV-infected subjects compared to controls (15). That report was in good correlation with our present findings. CD26 is present on various cell types, including T cells and epithelial cells of the liver, kidney, and intestine (33). DPPIV enzyme activity has been found in plasma, serum, and urine (34 –36) and levels in all these samples have been found to vary considerably with changes in immune status (37–39), in particular during autoimmune processes such as RA and SLE. However, the precise source and mechanism of the production of plasma sCD26/DPPIV remains unclear. The use of potential surrogate markers has been advocated for monitoring HIV disease progression, including CD4 1 T cells (expressed as an absolute number, a percentage of lymphocytes, or a ratio of CD4 1 to CD8 1 T cells), viral load (HIV-1 RNA), b 2-microglobulin, and neopterin (29, 40, 41). Of these markers, HIV-1 RNA and CD4 1 T cell count are generally accepted as the most reliable markers for monitoring HIV disease (41). Furthermore, it is recently established that plasma HIV-1 RNA determinations are the most important prognostic markers of disease progression and provide the most valuable tool for management of individual patients (29). Our cross-sectional and longitu-

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FIG. 5. Significant increase of specific DPPIV enzyme activity of plasma sCD26 associated with clinical improvement after anti-HIV therapy. Two representative patients (A: a 22-year-old asymptomatic female patient, B: a 26-year-old asymptomatic homosexual patient) were presented. Dotted lines on the left vertical axis indicate the lower limit of detection of plasma HIV-1 RNA determined by use of the Amplicor HIV Monitor kit (Roche Molecular Systems) (400 copies/ml).

dinal studies demonstrated that specific DPPIV enzyme activity was associated with HIV-1 RNA and CD4 count (Figs. 4 and 5), suggesting that specific DPPIV enzyme activity of sCD26 might be a potential surrogate marker and might play an important role in the immunopathogenesis of HIV disease. Recently, two CD26-like proteins, a 82-kDa DPPIV (DPPIV-b) and a 175-kDa DPPIV (DPPT-L), have been found (42, 43). The anti-CD26 mAbs used in this study

(1F7) does not react with DPPIV-b and only weakly reacts with DPPT-L. In contrast to 110-kDa CD26/ DPPIV, both proteins do not bind ADA. By using antiADA antibody, we were able to detect ADA in association with plasma sCD26 captured on an ELISA plate and more importantly, depletion of ADA-binding proteins by ADA-conjugated beads completely eliminated sCD26/DPPIV from plasma (data not shown). These findings suggest that the majority of plasma DPPIV

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TABLE 2 Correlation between Plasma sCD26/DPPIV Level and the Enhancing Effect of rsCD26 for PPD-Induced Lymphocyte Response in HIV-1 Infected Individuals Specific response a Subjects (CDC category)

CD4 count (/ml)

sCD26 (mg/ml)

Specific DPPIV enzyme activity (nmol/min/mg sCD26)

22.9 11.4 14.6 16.3 6 4.8

0.75 0.66 0.85 0.75 6 0.08

PPD 1 rsCD26 (cpm)

Enhancement index b

147 172 183

145 184 240

0.99 1.07 1.31 1.12 6 0.17

375 453 626 868

1059 1870 2407 3262

2.82 4.13 3.85 3.75 3.64 6 0.57

3934 4492 5105 6134

8727 6520 8619 9259

1.82 1.45 1.69 1.51 1.26 6 0.17

PPD (cpm)

Group 1 (subjects with CD4 count ,100) 1 (C) 2 (C) 3 (A)

56 34 23 mean 6 SD

Group 2 (subjects with CD4 count $100 and PPD response ,1000 cpm) 4 (A) 5 (B) 6 (A) 7 (B)

422 312 165 172 mean 6 SD

18.4 13.6 17.6 13.4 15.8 6 2.3

0.83 0.76 0.89 0.72 0.80 6 0.07 c

Group 3 (subjects with CD4 count $100 and PPD response $1000 cpm) 8 (B) 9 (A) 10 (A) 11 (B)

220 479 466 369 mean 6 SD

10.8 10.7 10.3 9.3 10.4 6 0.4 d

0.91 1.02 0.83 0.86 0.91 6 0.07

Concentrations of PPD for stimulation were 1 or 10 mg/ml, and rsCD26 was added at 1 mg/ml. PPD-specific response 5 (PPD cpm 2 cells alone cpm). PPD 1 rsCD26-specific response 5 [(PPD 1 rsCD26 cpm) 2 rsCD26 cpm]. b Enhancement index 5 (PPD 1 rsCD26-specific response)/(PPD-specific response). c P , 0.04 compared with uninfected controls (0.95 6 0.13, Fig. 1B) by Mann–Whitney U test. d P , 0.005 compared with uninfected controls (14.9 6 3.1, Fig. 1A) by Mann–Whitney U test. a

enzyme activity detected in this study was conventional 110-kDa CD26/DPPIV but not the novel form of serum 175-kDa DPPIV. It should be noted that the plasma concentration of sCD26 in this study was determined with recombinant 110-kDa sCD26 as a standard, and its specific DPPIV enzyme activity was calculated using this sCD26 level. Our present study also revealed that the plasma ADA level was significantly elevated in HIV-1 infected individuals and inversely correlated with specific DPPIV enzyme activity (Fig. 3). ADA plays a critical role in immune function through modulating the extracellular concentration of adenosine. It is well known that patients with congenital ADA deficiency develop a severe immunodeficiency (44). ADA is detected in plasma and is also present on the surface of CD26 1 T cells, as CD26 is the ADA binding protein (10). ADA activity in peripheral blood null lymphocytes from patients with AIDS has been shown to be elevated (45). Since a selective decline of CD26 1 T cells occurred in HIV-1 infected individuals (14, 15), elevated levels of serum ADA reported in AIDS patients (46) may be a consequence of the increasing destruction of CD4 1 T cells, especially CD4 1CD26 1 T cells.

Our previous study showed that PBLs from healthy individuals whose plasma sCD26 was high and could respond strongly to tetanus toxoid stimulation were insensitive to the enhancing effects of exogenously added sCD26 (11). In our present study, Group 3 (Table 2) showed a stronger response to PPD which was comparable to that of healthy individuals and minimal enhancing effects of sCD26, but significantly lower plasma sCD26 in comparison to healthy individuals. However, the highest specific DPPIV enzyme activity in the plasma of this group was observed among all three groups. Since DPPIV enzyme activity was the most important factor for enhancement of sCD26 on memory T cell response, in vivo high DPPIV enzyme activity could compensate for the low sCD26 levels in the plasma. Patients from Group 2 demonstrated higher levels of sCD26 than those of Group 3 although the specific DPPIV enzyme activity was lower than that of Group 3. This fact may partly explain why the addition of rsCD26 significantly restores the response to PPD in Group 2 and minimally in Group 3. CD26 may exert its costimulatory activity through binding with a putative ligand on the same or other cells and participate in

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the control of cellular functions through DPPIV enzyme processing of biologically active molecules. Thus, it is conceivable that decreased plasma DPPIV enzyme activity might in part explain the defective immune response in HIV-1 infected individuals. Our present study showed that proliferation of PPD-responsive T cells of even some HIV-1 infected individuals with CD4 1 T cell counts under 300/mm 3 could be enhanced by the addition of rsCD26. This is in contrast to the report of Schmitz et al. (47), who reported that early in HIV infection sCD26 serves as a decoy receptor for HIV-1 Tat and restores the defect in in vitro recall antigen response, but not in HIV-1 infected individuals with CD4 1 T cell counts under 300/mm 3 . Several possible explanations for these discrepancies include their use of Candida albicans as recall antigen and the source of sCD26 as a purified form from porcine and murine kidneys, rather than the PPD and rsCD26, respectively, used in the present study. It is known that DPPIV enzyme activity cleaves aminoterminal dipeptides with either L-proline or L-alanine at the penultimate position (5), which are found in many cytokines and chemokines. However, it is not yet clear whether most of these cytokines are physiological substrates of DPPIV. Recently, it has been reported that chemokine receptors, CXCR-4 and CCR-5, are co-receptors for T cell line-tropic HIV-1 (23) and macrophage-tropic HIV-1 (24, 25), respectively. CXCR-4 is the receptor for SDF-1 (stromal cell-derived factor-1) and CCR-5 is the receptor for RANTES (regulated-upon-activation, normal T expressed and secreted), MIP (macrophage inflammatory protein)-1a and MIP-1b (48). It has been reported that RANTES is a substrate for the DPPIV enzyme and the cleavage of RANTES by CD26 does not reduce or even increase the anti-HIV activity of RANTES (49, 50). In contrast, the cleavage of SDF-1 by CD26 reduces its anti-HIV activity and thus the presence of CD26 might facilitate the increasing HIV entry (51, 52). Taken together, our results strongly suggest that in vivo supplementation with rsCD26 in early stages of HIV disease may be of use not only for restoring the immunodeficiency state of HIV-1 infected individuals but also for delaying the progression of HIV disease. Further studies are in progress to define the role of DPPIV enzyme activity in HIV-1 infection. ACKNOWLEDGMENTS We thank Mrs. M. Gotoh for excellent technical supports and Dr. N. Satoh for kind help. We also thank P. J. Utz for review of the manuscript and L. Willis for typing the manuscript. This work is supported in part by grants from the Ministry of Education, Science, Sport, and Culture and the Ministry of Health and Welfare, Japan, and by the Program for Promotion of Fundamental Studies in Health

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Received September 15, 1998; accepted with revision February 18, 1999