Infection with Human T-Lymphotropic Virus Types I and ... - Europe PMC

CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, May 1995, p. 349–355 1071-412X/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 2, No. 3

Infection with Human T-Lymphotropic Virus Types I and II Results in Alterations of Cellular Receptors, Including the Up-Modulation of T-Cell Counterreceptors CD40, CD54, and CD80 (B7-1) CHARLENE S. DEZZUTTI,* DONNA L. RUDOLPH,

AND

RENU B. LAL

Retrovirus Diseases Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333 Received 8 November 1994/Accepted 9 February 1995

To examine the phenotypic alterations associated with human T-lymphotropic virus types I and II (HTLV-I and -II) infection, long-term cell lines (n 5 12 HTLV-I cell lines; n 5 11 HTLV-II cell lines; n 5 6 virus-negative cell lines) were analyzed for the cell surface expression of various lineage markers (i.e., myeloid, progenitor, and leukocyte), integrin receptors, and receptor-counterreceptor (R-CR) pairs responsible for cellular activation. As expected, all cell lines expressed the markers characterizing the leukocyte lineage (CD43, CD44, and CD53). Of the progenitor-myeloid markers examined (CD9, CD13, CD33, CD34, and CD63), only the percent expression of CD9 was significantly increased on HTLV-I and -II-infected cell lines as compared with that on virus-negative cell lines. Analysis of the b1 integrin subfamily (CD29, CD49b, CD49d, CD49e, and CD49f) showed no significant change, except that CD49e was significantly decreased on the HTLV-infected cell lines. For the b2 integrin subfamily, the cell surface density was increased for CD18 and CD11a, while the CD11c molecule was expressed exclusively on the HTLV-I- and HTLV-II-infected cell lines. Analysis of several R-CR pairs (CD2-CD58, CD45RO-CD22, CD5-CD72, CD11a-CD54, gp39-CD40, and CD28-CD80) demonstrated that comparable levels of expression of the Rs (CD2, CD45RO, CD5, and CD28) and of some of the CRs (CD58, CD22, and CD72) were in all cell lines; however, CD54, CD40, and CD80 were expressed constitutively on the HTLV-I- and HTLV-II-infected cell lines. Functionally, the expression of these R-CR pairs did not appear to affect the autologous proliferation, since monoclonal antibodies to these R-CR pairs were not able to inhibit proliferation of the infected cell lines. Taken together, our results indicate that HTLV-I and -II can modulate the expression of several T-cell activation molecules and CRs normally expressed on alternate cell types. activation. Studies have indicated that HTLV-I-infected peripheral blood lymphocytes (PBLs) and cell lines have altered expression of several lineage markers, specifically a myelomonocyte lineage marker, CD13, and a granulocyte and macrophage precursor marker, CD33 (11, 19–21). More recently, the expression of two CRs that have a wide tissue distribution on both hematopoietic and nonhematopoietic cell types, CD54 and CD58, was shown to be higher on HTLV-Iinfected PBLs and cell lines (9, 16). Both of these CRs along with their respective Rs (CD11a and CD2) are important costimulatory molecules involved in T-cell activation. Binding of CD2 by CD58 induces the alternative pathway of T-cell activation and proliferation (15), while the binding of CD11a by CD54 enhances the proliferation and production of cytokines in CD3-activated T cells (7, 10, 24). To obtain a comprehensive view of phenotypic alterations as well as their potential role in the activation and proliferation of infected cells, we have established HTLV-I- and HTLV-IIinfected cell lines derived from asymptomatic individuals as well as from patients with adult T-cell leukemia or HTLV-Iassociated myelopathy (5). We have examined lineage and integrin markers in addition to R-CR pairs responsible for cellular activation. Our findings indicate that the HTLV-I- and HTLV-II-infected cell lines are primarily of the leukocyte lineage, with altered expression of several molecules involved in cellular activation and adhesion. More importantly, we demonstrate constitutive expression of several CR molecules (CD54, CD40, and CD80) on the HTLV-I- and HTLV-IIinfected cell lines.

In the early 1980s, human T-lymphotropic virus type I (HTLV-I) was shown to be etiologically associated with both leukemic and neurologic diseases. Extensive epidemiologic studies now report that a small proportion (#5%) of individuals infected with HTLV-I progress to either adult T-cell leukemia or HTLV-I-associated myelopathy, whereas the majority of the infected individuals remain asymptomatic (25). The related retrovirus, HTLV-II, has not been conclusively associated with any disease entity; however, there are a few reports of HTLV-II-infected individuals with a HTLV-I-associated myelopathy-like illness (13, 17). While HTLV-I is endemic in Japan, the Caribbean, and certain parts of Africa, HTLV-II is endemic in several native amerindian populations as well as in injecting drug users in the United States and Europe (25). Both retroviruses are genetically similar and encode a transregulatory protein, tax, which is capable of trans-activating several cellular promoters whose products are associated with T-cell activation (30). While it has clearly been shown that cellular receptors involved in T-cell activation are up-regulated during infection with HTLV-I (32, 33, 38), little is known about the potential alterations of other cellular receptors, which are characteristic of various cell lineages, or the modulation of receptor-counterreceptor (R-CR) pairs, which are important for cellular

* Corresponding author. Mailing address: Retrovirus Diseases Branch, Centers for Disease Control and Prevention, 1600 Clifton Rd., MS G19, Atlanta, GA 30333. Phone: (404) 639-1024. Fax: (404) 6391174. 349

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MATERIALS AND METHODS

RESULTS

Study population. Long-term T-cell lines developed from PBLs from patients infected with either HTLV-I (n 5 12; cell lines 1742, 1996, 3669, 3614, FS, IR, EG, SP, A212, 1657, MT-2, and HuT102) or HTLV-II (n 5 11; cell lines H1B, H1H, H2A, H2D, H2E, Y03, Y06, Y17, G12.1, AI1050, and MoT) were included in this study. Demographics of the patients and characterizations of the cell lines were described previously (5). Additionally, transformed T-cell lines that were HTLV-I and HTLV-II negative were used as controls (n 5 6; cell lines A2.01 [8], A3.01 [8], HSB-2 [1], Jurkat [35], Molt3 [26], and Molt4 [26]). All cell lines were maintained in RPMI 1640 supplemented with 10 to 15% fetal bovine serum, 100 U of penicillin per ml, 100 mg of streptomycin per ml, and L-glutamine. The HTLV-I- and HTLV-II-infected cell lines were further supplemented with 1 to 10% interleukin-2 (IL-2; ABI, Columbia, Md.) (5). Immunofluorescence analysis. After the HTLV-infected cell lines were washed twice in phosphate-buffered saline (PBS), 50,000 cells were spotted onto a slide and allowed to adhere for 15 min at room temperature. The cells were fixed in cold acetone for 5 min and rehydrated in PBS. Excess water was removed from the slide, and 10% normal goat serum in PBS (blocking buffer) was overlaid on the cells for 15 min at room temperature. The blocking buffer was removed, and a 1:20 dilution of fluorescein isothiocyanate-labeled anti-HTLV polyclonal antibody (gift from S. McDougal, Centers for Disease Control and Prevention) was overlaid on the cells for 30 min at room temperature in the dark. The slide was subsequently washed four times in PBS. Cells expressing HTLV antigens were detected with a fluorescent microscope. Flow cytometric analysis. Phenotype analysis of the cell lines was performed by flow cytometry analysis with a FACScan (Becton Dickinson, San Jose, Calif.). Briefly, 50,000 cells were stained individually with monoclonal antibodies (MAbs) to the cluster of differentiation (CD) antigens for 30 min at 48C. The MAbs were conjugated with either fluorescein isothiocyanate or phycoerythrin or left unlabeled and required the addition of a fluorescein isothiocyanate-labeled secondary antibody. The MAbs to CD2 (S5.2), CD3 (SK7), CD5 (L17F12), CD45RO (UCHL1), CD19 (4G7), CD11c (S-HEL-3), CD80 (L307.4), CD58 (AICD58), CD25 (2A3), FCD71 (L01.1), and HLA-DR (L243) were obtained from Becton Dickinson (Mountain View, Calif.); MAbs to CD28 (CD28.2), CD21 (BL13), CD33 (D3HL60.251), CD34 (QBEND10), CD44 (J173), CD63 (CLBGran/12), CD49b (Gi9), CD49d (HP2.1). CD49e (SAM1), CD49f (GoH3), CD18 (BL5), CD11a (25.3), CD11b (Bear-1), CD61 (S221), and CD54 (84H10) were obtained from AMAC, Inc. (Westbrook, Maine); MAbs to CD72 (Bu40), CD40 (B-B20), CD22 (RFB4), CD9 (MM2/57), CD53 (MEM53), and CD37 (WR17) were obtained from Harlin Bioproducts for Science (Indianapolis, Ind.); MAbs to CD13 (WM-47), CD20 (B-Ly1), and CD43 (DF-T1) were obtained from DAKO Corp. (Carpinteria, Calif.); and MAb to CD29 (4B4-RD1) was obtained from Coulter Immunology (Hialeah, Fla.). The cells were washed twice in cold buffer (PBS with 0.2% sodium azide, 0.1% bovine serum albumin [BSA], and 2% human antibody serum) and fixed in 1% paraformaldehyde for 30 min. The fixed cells were analyzed with a FACScan (Becton Dickinson), and data analysis was performed with Consort 30 software on a Hewlett-Packard computer. RNA purification and RT-PCR. Analysis of the CD3d rearrangement was performed by reverse transcriptase (RT)-PCR. RNA from each cell line was CsCl gradient purified and converted to cDNA with an RT buffer (53 RT buffer is 250 mM Tris HCl (pH 8.3), 30 mM MgCl2, 200 mM KCl, 50 mM dithiothreitol, and 0.05% Nonidet P-40), 20 ng of oligo(dT) per ml, 100 ng of BSA per ml, 0.5 mM each deoxynucleoside triphosphate (dNTP), 0.2 U of RNasin per ml, and 0.01 U of avian myeloblastosis virus RT per ml, brought to 50 ml with diethylpyrocarbonate-treated H2O. The samples were incubated at 428C for 2 h, after which the cDNA was precipitated, washed, and resuspended with H2O. PCR was performed on the samples by adding the cDNA to the PCR mixture (13 PCR buffer [Perkin-Elmer, Norwalk, Conn.], 0.6 mM each dNTP, 2.5 U of Taq polymerase [Perkin-Elmer], 200 ng of 59 and 39 primers per ml). The cDNA was amplified first with b-actin primers (59 primer 59-GTGGGGCGCCCCAGGC ACCA-39 and 39 primer 59-CTCCTTAATGTCACGCACGATTTC-39) to determine if there was amplifiable material. The same cDNA was then amplified with CD3d primers (59 primer 59-CTGGACCTGGGAAAACGCATC-39 and 39 primer 59-GTACTGAGCATCATCTCGATC-39). Both amplicons were separated on a 1.5% agarose gel. The CD3d gel was then blotted onto a nylon membrane (Hybond; Amersham Corp., Arlington Heights, Ill.) and probed with an internal end-labeled probe, 59-GCCGACACACAACTCTGTTGAGGA-39. After being washed, the blot was exposed to X-ray film. Role of R-CR interactions. To determine if R-CR interactions were important for continuous proliferation of HTLV-infected cell lines, two representative HTLV-I (MT-2 and 1996)- and HTLV-II (G12.1 and Y17)-infected cell lines were grown in 96-well plates in the absence or presence of various concentrations (1, 0.5, and 0.25 mg/ml) of MAbs against CD2, CD58, CD11a, CD54, CD11c, CD40, and CD80 for 7 days. During the last 18 h, the cells were pulsed with 0.5 mCi of [3H]thymidine per well. The plates were harvested, and the incorporation of thymidine was measured by liquid scintillation spectroscopy. Statistical analysis. Statistical analysis was performed with Student’s t test. The data are expressed as means 6 standard errors of the means.

HTLV-infected cell lines are derived from the leukocyte lineage and modulate integrin molecules. To characterize the expression of lineage markers on the HTLV-I- and HTLV-IIinfected cell lines, antibodies directed against progenitor, myeloid-monocyte, and leukocyte markers as well as integrin receptors were examined (Table 1). The expression of the myeloid-monocyte marker CD9 was significantly increased (P , 0.05) on both HTLV-I-infected (85.3% 6 3.6%) and HTLVII-infected (96.2% 6 2.3%) cell lines when compared with that of the virus-negative cell lines (28.3% 6 15.8%). The myelomonocyte precursor CD13 was minimally expressed on 5 of 12 HTLV-I-infected cell lines (1742, 3614, EG, SP, and 1657) and on 1 of 11 HTLV-II-infected cell lines (MoT) and was undetectable on the virus-negative cell lines. Of the remaining progenitor or myeloid markers, there was no difference in the expression of CD33, CD34, and CD63 on any of the cell lines examined. The leukocyte markers CD43, CD44, and CD53 were expressed on greater than 70% of the cells for each cell line examined, suggesting that both HTLV-I and -II cell lines were of leukocyte lineage (Table 1). Because integrin molecules increase adhesion of PBLs to endothelial cells and antigen-presenting cells and can influence cellular activation (7, 10, 31), we examined the expression of b1, b2, and b3 integrin subfamilies on HTLV-I- and HTLVII-infected cell lines. Within the b1 integrin subfamily, levels of expression of CD29 (b1), CD49b (a2), CD49d (a4), and CD49f (a6) were similar on the three groups of cell lines examined (Table 1). Interestingly, the expression of CD49e (a5) was decreased significantly (P , 0.001) on both HTLVI-infected (26.1% 6 10.5%) and HTLV-II-infected (50.7% 6 8.0%) cell lines when compared with that of the virus-negative cell lines (94.7% 6 2.6%) (Fig. 1; Table 1). The percentage of cells expressing the b2 integrin markers CD18, CD11a, and CD11b did not vary in the three groups of cell lines (Table 1); however, the cell surface densities of CD18 (Fig. 1) and CD11a (LFA-1) receptors were increased on both HTLV-I- and HTLV-II-infected cell lines. While no expression of CD11c was detected on the virus-negative cell line group, virtually all of the HTLV-I- and HTLV-II-infected cell lines expressed CD11c (Table 1; Fig. 1). Of the b3 integrin subfamily, minimal expression of CD61 (b3) was detected on the three groups of cell lines (Table 1). HTLV-infected cell lines primarily express T-cell markers. Further examination of these cell lines for B-cell markers showed no expression of CD19 or CD37 on any cell line. Minimal expression of CD20 was detected on HTLV-I- and HTLV-II-infected cell lines. Five of 12 HTLV-I-infected (3669, 3614, A212, HuT102, and MT-2) and 3 of 11 HTLV-IIinfected (Y17, G12.1, and MoT) cell lines expressed moderate to high amounts of CD21 (22 to 100%), while none of the virus-negative cell lines expressed CD21 (Table 2). The levels of expression of T-cell lineage markers (CD2, CD3, CD5, CD28, and CD45RO) were similar among the virus-negative cell lines and the HTLV-I- and HTLV-II-infected cell lines (Table 2). To ensure that these cell lines were derived from the T-cell lineage, RT-PCR was performed with primers that amplify the mRNA of the CD3d rearrangement. All of the cell lines examined were positive (Table 3), indicating that they were derived from T cells. Further analysis of selected cell lines reveals that they can be placed into three groups on the basis of the percentage of virus-expressing cells. The high (.75%) virus expressers represent those cell lines that have been in culture for more than 10 years and are not dependent on IL-2 (HuT102, MT-2, and MoT), while the mod-

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TABLE 1. Expression of leukocyte markers on HTLV-infected cell lines % Positive cells (mean 6 SEM) CD designation

Progenitor or myeloid CD9 CD13 CD33 CD34 CD63 Leukocyte CD43 CD44 CD53 Integrin receptors CD29 CD49b CD49d CD49e CD49f CD18 CD11a CD11b CD11c CD61 a b

Cellular marker(s) Uninfected

HTLV-I infected

HTLV-II infected

Platelets, pre-B cell Myeloid Monocyte, macrophagegranulocyte progenitor Hematopoietic progenitor Activated platelets-monocytes, lysosomal membrane

28.3 6 15.8 0.0 0.0

85.3 6 3.6a 41.2 6 13.6a 11.3 6 8.4

96.2 6 2.3 20.2 6 15.8 0.0

0.0 54.7 6 4.4

0.0 42.3 6 8.6

0.0 35.3 6 8.0

T-cell-granulocyte-pre-B cell (leukosialin) T-cell-granulocyte-erythrocyte (homing receptor) Panleukocyte

99.2 6 0.7

98.4 6 1.2

95.2 6 3.1

97.8 6 2.0

99.9 6 0.1

85.6 6 8.3

97.8 6 0.9

93.8 6 2.8

94.1 6 1.6

b1, leukocytes b1a2, collagen receptor b1a4, fibronectin receptor b1a5, fibronectin receptor b1a6, laminin receptor b2, leukocytes LFA-1, leukocytes Mac-1, monocytes gp150-95, monocytes b3, platelets

98.3 6 1.3 29.7 6 20.3 94.7 6 2.6 94.7 6 2.6 0.0 42.3 6 19.3 79.8 6 14.0 28.3 6 15.8 0.0 11.2 6 6.6

99.5 6 0.2 41.0 6 9.3 98.1 6 0.9 26.1 6 10.5b 9.1 6 7.4 80.6 6 6.9 90.2 6 7.7 36.0 6 7.4 60.8 6 7.5 14.7 6 6.2

98.3 6 1.3 13.3 6 4.7 93.6 6 2.8 50.7 6 8.0b 0.0 71.1 6 9.8 83.9 6 9.6 36.0 6 23.5 69.9 6 9.7a 25.5 6 9.8

P , 0.05 in comparison with virus-negative cell line. P , 0.001 in comparison with virus-negative cell line.

erate (26 to 74%) virus expressers (1996 and 3669) and low (,25%) virus expressers (FS, A212, H2A, and H2D) have been in culture for approximately the same time but vary in their need for IL-2 supplementation. The moderate virus ex-

pressers required 0 to 1% IL-2, and the low virus expressers required 10% IL-2. Regardless of their virus expression, all of the HTLV-I- and HTLV-II-infected cell lines expressed CD25 (IL-2 receptor), CD71 (transferrin receptor), and HLA-DR,

FIG. 1. Expression of integrin molecules on HTLV-I- and HTLV-II-infected cell lines. The determination of expression of CD29, CD49e, CD18, and CD11c was performed as described previously. Three representative cell lines (A3.01 [negative] [dotted line], 1996 [HTLV-I] [bold line], and G12 [HTLV-II] [thin line]) are shown. MFC, mean fluorescence channel.

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TABLE 2. Expression of T- and B-cell lineage markers on HTLV-infected cell lines % Positive cells (mean 6 SEM)

CD designationa

Cellular markers

CD19 CD20 CD21 CD37 CD2 CD3 CD5 CD28 CD45RO

Pan-B cell Pre-B cell Mature B cell B cell T cell T cell T-cell or B-cell subpopulation T cell Memory T cell

a b

TABLE 3. T-cell CR expression on HTLV-infected cell lines Cell line

PCR-confirmed HTLV-I 1742 1996 3669b 3614 FS IR EG A212 SPb 1657 HuT102 MT-2 PCR-confirmed HTLV-II AI1050 H1B H1H H2A H2D H2E Y03 Y06 Y17 G12.1 MoT Virus negative A2.01 A3.01 HSB-2 Jurkat Molt3 Molt4 b

HTLV-I infected

HTLV-II infected

0.0 0.0 0.0 0.0 74.8 6 14.9 31.5 6 18.8 98.2 6 0.7 39.3 6 22.6 50.3 6 19.5

0.0 12.7 6 5.6 62.7 6 15.0b 0.0 92.6 6 6.3 76.5 6 12.3 91.4 6 8.0b 53.6 6 14.4 83.1 6 9.4

0.0 4.2 6 1.7 16.8 6 7.4 0.0 99.3 6 0.3 88.0 6 10.8 87.1 6 3.3b 52.7 6 13.8 99.0 6 0.5

B and T cells. P , 0.05 in comparison with virus-negative cell lines.

while the virus-negative cell lines had no detectable expression of these markers (data not shown). Several T-cell CRs are expressed on HTLV-infected cell lines. Recent studies have shown that effective activation of T cells requires at least one costimulatory signal delivered via the engagement of certain T-cell surface accessory molecules (i.e., Rs) by their appropriate CRs expressed on the surface of antigen-presenting cells. These R-CR pairs include CD2CD58, CD5-CD72, CD11a-CD54, CD45RO-CD22, gp39-

a

Uninfected

CD3d rearrangementa

% Positive cells (mean 6 SEM) CD40

CD54

CD80

1 1 1 1 ND ND 1 1 1 1 1 1

4 74 98 100 0 4 6 100 0 87 93 98

91 60 100 100 90 64 56 100 99 100 100 100

100 98 99 95 95 99 100 100 97 97 100 100

ND 1 1 1 1 1 1 1 1 ND 1

13 52 33 22 0 2 14 15 44 89 100

80 60 45 48 71 74 82 67 75 99 99

80 72 100 85 98 93 99 92 97 96 99

1 1 1 1 1 ND

ND 0 0 0 0 0

6 2 73 6 0 0

0 6 5 0 0 0

ND, not determined; 1, present. SP and 3669 were grown in medium without exogenous IL-2.

CD40, and CD28- or CTLA-4-CD80. We have already shown that most of the Rs remain unchanged in the HTLV-infected cell lines (Tables 1 and 2). We next examined whether infection with HTLV-I or HTLV-II could modulate the expression of their respective CRs. While no difference in the expression of CD58 was detected among the three groups of cell lines, cell surface expression of CD40, CD54, and CD80 was higher on the HTLV-infected cell lines when compared with that of virus-negative cell lines (Fig. 2). A detailed analysis of all cell lines demonstrated that 7 of 12 HTLV-I-infected cell lines and 6 of 11 HTLV-II-infected cell lines expressed high levels of CD40 (Table 3). All of the HTLV-I- and HTLV-II-infected cell lines expressed CD54 and CD80; among the virus-negative cell lines, only HSB-2 expressed CD54 (Table 3). The cumulative analysis of the cell lines for various CRs is shown in Fig. 3. While no significant differences were observed for CD22, CD58, and CD72, the levels of expression of CD40, CD54, and CD80 were significantly increased (P , 0.001) in both HTLVI-infected (73.1% 6 12.7%, 88.3% 6 5.1%, and 98.3% 6 0.6%, respectively) and HTLV-II-infected (38.4% 6 10.5%, 72.7% 6 5.3%, and 95.8% 6 1.0%, respectively) cell lines when compared with their levels of expression in virus-negative cell lines (0.0%, 17.6% 6 13.9%, and 5.5% 6 0.5%, respectively). Furthermore, the levels expression of CD40, CD54, and CD80 were independent of the level of viral expression because cell lines with low, moderate, or high viral expression had equivalent levels of expression of these CRs. The importance of the expression of these molecules for cellular proliferation or activation was assessed by blocking the interaction of these R-CR pairs with MAb. Two HTLV-infected cell lines from each group (MT-2 and 1996 [HTLV-I]; G12.1 and Y17 [HTLV-II]) that expressed high levels of CD11a, CD11c, CD54, CD40, and CD80 were used. None of the MAbs against these markers inhibited the proliferation of these cell lines (data not shown). Similar concentrations of CD11a and CD54 inhibited the spontaneous proliferation of PBLs from HTLV-II-infected persons (4). These data suggest that proliferation of immortalized HTLV-infected cell lines is not directly dependent on activation through these R-CR interactions. DISCUSSION Infection with HTLV-I or HTLV-II not only leads to T-cell immortalization but also results in alterations in cell surface phenotypes. While several studies have analyzed cell surface expression of isolated markers by use of HTLV-I-infected PBLs and cell lines (9, 11, 16, 19–21), a comprehensive analysis

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FIG. 2. Exclusive expression of several T-cell CRs on HTLV-I- and HTLV-II-infected cell lines. The levels of expression of CD58, CD40, CD54, and CD80 are shown for three representative cell lines (A3.01 [negative] [dotted line], 1996 [HTLV-I] [bold line], and G12 [HTLV-II] [thin line]). MFC, mean fluorescence channel.

of these markers has not been performed. More importantly, little is known regarding cell surface modulation following HTLV-II infection. Therefore, we have phenotypically characterized 12 HTLV-I-infected and 11 HTLV-II-infected cell lines for the expression of several lineage-specific markers, integrin receptors, and R-CR pairs found to be necessary for activation and proliferation of T cells. Our results indicate that infection with either HTLV-I or HTLV-II results in the modulation of

FIG. 3. Cumulative analysis of expression of several CRs showing exclusive expression of CD40, CD54, and CD80 on HTLV-infected cell lines. Virusnegative (s), HTLV-I-infected (o), and HTLV-II-infected (■) cell lines were assessed for their expression of CD58, CD22, CD72, CD54, CD40, and CD80. The levels of expression of CD40, CD54, and CD80 were significantly higher on the HTLV-infected cell lines than on the virus-negative cell lines (*, P , 0.05; **, P , 0.001). No difference in the levels of expression of CD58, CD22, and CD72 was observed.

similar markers, specifically those important for cellular activation and proliferation. Of the several myeloid family markers studied, an increase in the expression of CD9 was observed on all HTLV-I- and HTLV-II-infected cell lines. CD9 is expressed primarily on activated platelets and can also be detected on pre-B cells and on activated T cells but not on resting mature T cells or B cells (3). The increased expression of CD9 detected on HTLVinfected cells may therefore reflect their activated state. The expression of CD13 was detected on a few HTLV-I- and HTLV-II-infected cell lines, while CD33 and CD34 were minimally expressed on the HTLV-I- and HTLV-II-infected cell lines. These data are in contrast to those of previous studies which showed CD13 expression on all of the HTLV-I-infected cell lines and CD33 expression on 12 of 19 HTLV-I-infected cell lines (11, 19–21). The detection of CD13 and CD33 in the previous studies may be due to the relatively short time in culture for their cell lines (,5 months) as compared with that of the cell lines used in this study (.2 years), and our findings suggest that the expression of CD13 and CD33 markers may be an early transient event. As expected, all of the HTLV-I- and HTLV-II-infected cell lines were shown to be of the leukocyte lineage, and they expressed CD43, CD44, and CD53. Taken together, these data suggest that HTLV-I and HTLV-II primarily infect the leukocyte lineage and appear to have minimal modulatory effect on other lineage markers. Analysis of the integrin subfamilies on HTLV-I- and HTLVII-infected cell lines demonstrated altered cell surface expression. Within the b1 subfamily, the expression of CD29, CD49b, CD49c, CD49d, and CD49f did not vary among the three groups of cell lines tested. We have demonstrated previously that the expression of CD29 is increased on spontaneously proliferating lymphocytes from HTLV-I- and HTLV-II-infected individuals when compared with CD29 expression on

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lymphocytes from control individuals (4, 23). This discrepancy may reflect the high level of CD29 expressed on transformed T cells in general (i.e., the high expression of CD29 on virusnegative cell lines) as compared with that on normal PBLs. Interestingly, CD49e expression was decreased on both infected cell line groups. In vitro infection of PBLs with HTLV-I has been shown previously to lead to the induction of CD49d and CD49e early during infection, but the data for the stably transformed cell lines were not reported (6). The induction of CD49d and CD49e therefore may result from an acute infection with HTLV-I and may not necessarily reflect the stably transformed phenotype. For the b2 integrin subfamily, the relative levels of expression of CD11a and CD18 (LFA-1) remained similar among the three groups of cell lines tested; however, the cell surface density (the number of receptors on the cells) of these markers was increased on the HTLV-I- and HTLV-II-infected cell lines. These data corroborate our previous findings that demonstrated increased cell surface density of CD11a or CD18 on spontaneously proliferating lymphocytes from HTLV-II-infected individuals (4). The CD11c marker, normally found on monocytes, granulocytes, and certain subsets of activated T cells, was expressed exclusively on HTLV-Iand HTLV-II-infected cell lines and may represent the activated state of the HTLV-infected cells. Within the leukocyte lineage, all of the cell lines were shown to be of T-cell origin. As expected, both the HTLV-I-infected cell lines and the HTLV-II-infected cell lines expressed several T-cell activation markers (CD25, CD71, and HLA-DR) (32, 33, 38). In agreement with a previous study, none of the HTLV-infected cell lines expressed any of the B-cell markers (11). Interestingly, CD21, a receptor for the Epstein-Barr virus, was detected on several of the HTLV-infected cell lines. PCR analysis with oligoprimers specific for the Epstein-Barr virus genome demonstrated that all of the HTLV-infected cell lines were negative for Epstein-Barr virus (data not shown). While T-cell activation and proliferation occur predominantly through the interaction of the T-cell receptor–CD3 complex and major histocompatability complex-antigen, recent studies have shown that several costimulatory Rs also need to bind to their appropriate CRs on antigen-presenting cells (CD2-CD58, CD5CD72, CD11a-CD54, CD28-CD80, CD45RO-CD22, and gp39CD40) for complete T-cell activation (29). Analysis of the cell lines revealed little differences in the expression of CD2, CD3, CD5, CD28, and CD45RO between the HTLV-infected and virus-negative cell lines. This result is to be expected since all three groups of cell lines studied are T cell derived as confirmed by the detection of the CD3d rearrangement. However, a selective expression of several of the CRs was observed; CD40 and CD80 as well as CD54 were expressed exclusively on the HTLV-I- and HTLV-II-infected cell lines. Cell surface expression of CD54 and CD80 has been demonstrated previously on a limited number of HTLV-I-infected cell lines (9, 34). Of these R-CR pairs, CD2-CD58 and CD11a-CD54 have been shown previously to be important for HTLV-I- and HTLV-II-associated proliferation of uninfected PBLs, and this interaction was shown to be independent of virus antigen production (4, 14, 18, 36). To determine what role these R-CR pairs and others may have in autologous proliferation of the HTLV-infected cells, we attempted to block the proliferation of several HTLV-I- and HTLV-II-infected cell lines expressing CD2-CD58, CD11a-CD54, CD28-CD80, and gp39-CD40 with specific MAbs. These R-CR pairs were chosen not only because they have been shown previously to inhibit HTLV-associated proliferation (CD2-CD58 and CD11a-CD18) (4, 18, 36) but also because of their importance in cellular activation


(CD28-CD80 and gp39-CD40). None of the MAbs directed against these R-CR pairs inhibited the autologous proliferation of these cell lines. These data imply that the expression of these R-CR pairs do not appear to be required for autologous proliferation of these infected cell lines, and other factors such as the transactivating protein tax may play a role. Indeed, tax has been shown not only to modulate the cell surface expression of CD54 (9) but also to activate the CD54 promoter (27, 28). It is of further interest to note that the C8166-45 cell line, which contains several defective HTLV-I proviral genomes and expresses only a functional tax protein (2), expresses not only CD54 on its surface but also CD40 and CD80 (data not shown). In conclusion, we have shown the increased expression of cellular markers on the HTLV-I- and HTLV-II-infected cell lines. These markers are normally found on platelets (CD9) and myeloid cells (CD11c), but they are also detected on subpopulations of activated T cells. Additionally, infection by HTLV-I or HTLV-II leads to the up-modulation of several CRs normally expressed on antigen-presenting cells (CD40, CD54, and CD80). Whether the expression of these antigens is a direct or indirect result of infection is unknown at this time and is being investigated currently. While the expression of these R-CR pairs does not appear to affect the proliferation of the immortalized cell lines, their expression clearly influences the proliferation of uninfected lymphocytes (18, 22). Furthermore, we and others have shown that HTLV-I- and HTLV-IIinfected cell lines and human immunodeficiency virus-infected cells lines have the ability to act as antigen-presenting cells and that this activity can be blocked with MAbs against CD11a, CD54, CD58, and CD80 (12, 22, 37). The involvement of these R-CR pairs warrants further study for a defined role in HTLVassociated proliferation. ADDENDUM IN PROOF We have recently screened several HTLV-1-infected (MT-2 and HuT102) and HTLV-II-infected (MoT AI1050) cell lines for the expression of CD86 (B7-2). Both HTLV-I- and HTLVII-infected cell lines had 100% expression of CD86, while no expression was detected on the uninfected cell line (A3.01). These data provide further proof that infection with HTLV-I or HTLV-II results in the aberrant expression of T-cell CRs. REFERENCES 1. Adams, R. A., A. Flowers, and B. J. Davis. 1968. Direct implantation and serial transplantation of human acute lymphoblastic leukemia in hamster, SB-2. Cancer Res. 28:1121–1125. 2. Bhat, N. H., Y. Adachi, K. P. Samuel, and D. Derse. 1993. HTLV-I gene expression by defective proviruses in an infected T-cell line. Virology 196: 15–24. 3. Boucheix, C., P. Benoit, P. Frachet, M. Billard, R. E. Worthington, J. Gagnon, and G. Uzan. 1991. Molecular cloning of the CD9 antigen: a new family of cell surface proteins. J. Biol. Chem. 5:117–122. 4. Dezzutti, C. S., D. L. Rudolph, S. Dhawan, and R. B. Lal. 1994. Modulation of HTLV-II-associated spontaneous lymphocyte proliferation by b2 integrin CD11a/CD18 involves interaction with its cognate ligand, CD54. Cell. Immunol. 156:113–123. 5. Dezzutti, C. S., D. L. Rudolph, C. R. Roberts, and R. B. Lal. 1993. Characterization of human T-lymphotropic virus type I- and II-infected T-cell lines: antigenic, phenotypic, and genotypic analysis. Virus Res. 29:59–70. 6. Dhawan, S., B. S. Weeks, F. Abbasi, H. R. Gralnick, A. L. Notkins, M. Klotman, K. M. Yamada, and P. E. Klotman. 1993. Increased expression of a4b1 integrins on HTLV-I-infected lymphocytes. Virology 197:778–781. 7. Fan, T.-S., A. A. Brian, B. A. Lollo, N. Mackman, N. L. Shen, and T. S. Edgington. 1993. CD11a/CD18 (LFA-1) integrin engagement enhances biosynthesis of early cytokines by activated T cells. Cell. Immunol. 148:48–59. 8. Folks, T., S. Benn, A. Rabson, T. Theodore, M. D. Hoggan, M. Martin, M. Lightfoote, and K. Sell. 1985. Characterization of a continuous T-cell line susceptible to the cytopathic effects of the acquired immunodeficiency syndrome (AIDS)-associated retrovirus. Proc. Natl. Acad. Sci. USA 82:4539–4543.

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