The role of CD40 and CD80 accessory cell molecules in dendritic cell-dependent HIV-1 infection

Immunity, Vol. 1, 317-325,

July, 1994, Copyright 0

1994 by Cell Press

The Role of CD40 and CD80 Accessory Cell Molecules in Dendritic Cell-Dependent HIV-1 Infection Lesya M. Pinchuk,‘? Patricia S. Polacino,‘t Michael B. Agy,‘t Stephen J. Klaus,* and Edward A. Clark,‘tS *Regional Primate Research Center TDepartment of Microbiology *Department of Immunology University of Washington Medical Center Seattle, Washington 98195

Summary We investigated the role of blood dendritic cells (DCs) in transmission of HIV-1 from infected to uninfected CD4+ T cells, and the accessory molecules involved. DCs promoted transmission from infected to uninfected CD4+ cells, but DCs themselves were not infectable. DC-mediated transmission was blocked by MAb to CD4 and MHC class II, but strongly increased by MAb to CD40 on DCs or CD28 on T cells. The DCdependent infection was inhibitable by anti-CD80 and a soluble fusion protein of the CD80 ligand, CTLA4; soluble CTLA4 immunoglobulin also blocked infection augmented by cross-linking CD40. These data suggest a linkage between CD40-CD40L and CD28-CD80 counterreceptors on DCs and T cells, and spread of HIV infection in vivo.

al., 1992). Cross-linking CD40 on B cells by antibody or activated T cells induces expression of CD80 (Ranheim and Kipps, 1993). The CD40L-CD40 and CD28-CD80 receptor-ligand pairs appear to play a key role in the cognate T-B dialog and to allow T cells and B cells to sense the state of their partners activation and then respond appropriately (Clark and Ledbetter, 1994). Whether HIV-1 can replicate in primary blood DCs is controversial. Initial studies found that normal DCs were susceptible to HIV-1 infection in vivo and in vitro and that HIV infection blocked APC function (Knight et al., 1993; Langhoff and Haseltine, 1992; Langhoff et al., 1991,1993; Macatoniaet al., 1989,1991; Patterson and Knight, 1987). DC cultures also supported much more virus production than did cultures of primary unseparated T cells, CD4+ T cells, and monocytes (Langhoff and Haseltine, 1992; Langhoff et al., 1991). However, other groups did not detect HIV-1 infection of DCs, and after exposure to HIV-l, DCs continued to present normal antigens and superantigens to T cells and drive T cell replication (Cameron et al., 1992a, 1992b; Weissman et al., personal communication). Here we investigated the role of DCs in the transmission of HIV-1 infection from infected CD4+ T lymphocytes to uninfected CD4+ cells and the involvement of CD40LCD40 and CD80-CD28 interactions in DC-dependent HIV replication. Results and Discussion

Introduction Dendritic cells (DCs) are highly efficient antigenpresenting cells (APCs) that initiate immune responses to peptide antigens associated with class II MHC (Freudenthal and Steinman, 1990; Macatonia et al., 1989; Steinman et al., 1993; Young and Steinman, 1987). DCs are extremely effective APCs because they express not only high levels of MHC class I and class II products but also several of the known accessory molecules. For example, some DCs express B7/BBl, now designated CD80 (Schlossman et al., 1994), a ligand for CD28 and CTLA4 on T cells. Expression of CD80 on blood DCs increases after in vitro culture (Thomas et al., 1993a), binding to allogeneic T cells (Younget al., 1992)or by culture with IFNyorgranulocytemacrophage colony-stimulating factor (GM-CSF) (Hart et al., 1993). Activated T cells are stimulated to proliferate and produce interleukin 2 (IL-2) by CD80 cross-linking of CD28 (Linsley and Ledbetter, 1993). Expression of CD80 plays an important role in the enhanced accessory function of cultured DCs for fresh CD4+ T cells (Thomas et al., 1993a). DCs also constitutively express the B cell-associated marker CD40 (Freudenthal and Steinman, 1990; Steinman et al., 1993). This is of interest since the ligand for CD40 (CD40L) is expressed on activated T cells (Armitage et al., 1992; Hollenbaugh et al., 1992) and provides a key helper Tcell signal to B lymphocytes(Armitage et al., 1992; Hollenbaugh et al., 1992; Noelle et al., 1992; Spriggs et

Purified DCs Are CD40+ and Are Potent Stimulators in Allogeneic Mixed Leukocyte Reaction Two approaches were used to document the enrichment of DCs. First, using flow cytometry, we found that purified DCs were not bound by a cocktail of monoclonal antibodies (MAbs) to CD3, CD1 4, CD1 6, or CD20, but as expected were CD40’ (Figure 1A). The detected purity of the DCs after cell sorting was up to 98%, consistent with previous studies (Weissman et al., personal communication). Analysis with the more sensitive FACScan revealed that the resulting population was 850/o-90% DCs, 1.5%-13% B cells, 2%-6% monocytes, l%-2% T lymphocytes, and O%-0.8% natural killers (see Experimental procedures). The resulting population of DCs was compared with monocytes and B cells by testing their ability to stimulate allogeneic T lymphocytes in primary mixed leukocyte reaction (MLR). Monocyte, B, and T cell populations were significantly less stimulatory than DCs (Figure 1 B). Purified DCs Are not Susceptible to HIV-l Infection Compared with Stimulated CD4+ T Cells or Unstimulated Peripheral Blood Mononuclear Cells To test whether DCs were susceptible to HIV-1 infection, we used polymerase chain reaction (PCR) with HIV-lspecific primers to compare HIV-1 DNA levels in APCs relative to control, peripheral blood mononuclear cells (PBMC), or activated CD4+ T cells. After a 16 hr exposure

Immunity 318

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to HIV-I, both CD4+ T cells activated by staphylococcal enterotoxin B and unseparated PBMC had high levels of viral DNA (Figure 2, lanes 5 and 6). In contrast, purified DCs and B cells exposed to HIV-1 had background levels of viral DNA comparable to T cells cultured with heatinactivated (HI) virus (Figure 2, lanes 3 and 4). These data confirm that.of Cameron et al. (Cameron et al., 1992b), who found that after exposure to HIV-l, stimulated CD4+ T lymphocytes but not purified DCs contained detectable proviral DNA. However, our results do differ from reports suggesting blood DCs are readily infected by HIV-1 (Langhoff et al., 1991, 1993; Patterson and Knight, 1987); Possible differences between our study and others include the method of preparing DCs (Cameron et al., 1992b; Langhoff et al., 1991; Patterson and Knight, 1987); heterogeneity in expression of key surface markers such as CD80 (Hart et al., 1993) and CD4, i. e., phenotypical and functional heterogeneity in the DC populations (Thomas et al.,

Figure 2. Purified DCs Are not Susceptibie to HIV-l pared with Stimulated CD4+ T Cells or Unstimulated

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The indicated cell populations were exposed to HIV-1 for 16 hr at 37% Control T cells were exposed to HI HIV-I. Cells were washed and DNAs were prepared for PCR using gag-specific sequences (primers SK38 and SK39). Lane 1, uninfected T lymphocytes; lane 2, T cells exposed to Hi HIV-l; lane 3, purified DCs; lane 4, CDlS+ B cells: lane 5, CD4+ T cells activated by the superanigen, staphylococcal enterotoxin B; lane 6, PSMC. Shown below are globin controls.

1993b); attachment time for infection; and the multiplicity of the input virus (Knight et al., 1993). DCs Promote HIV-1 Transmission from infected to Uninfected CD4+ T Cells To investigate how cell-cell interactions may influence HIV-1 production in T lymphocytes, we cultured infected CD4+ T lymphocytes with uninfected CD4+ T cells (see Experimental Procedures) in combination with 5 x lo4 autologous DCs (Figures 3A and 3D), monocytes (Figure 3B), or B cells (Figure 3C). CD4+ T cells incubated with DCs consistently displayed increased levels of HIV-1 DNA relative to T cells cultured alone (Figures 3A and 3D, lane 3; see Figure 4A, lane 5; see Figure 48, lane 4; see Figure 5, lane 4) which was totally blocked by MAb to CD4 (Figure 3, lane 4). Under these same conditions, monocytes and B cells were much less effective at augmenting HIV-I transmission (Figure 38, lane 5; Figure 3C, lane 4). In all experiments, when CD4+ T cells exposed to HIV-1 for 30

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min were added to uninfected CD4+ T cells, no viral DNA was detectable, showing the dependence on DCs for viral transmission. Levels of viral protein (~24) were measured in some experiments and paralleled results with PCR. For example, in two experiments, cultures with DCs augmented full viral replication to a mean of 597.1 pglml p24 viral protein (day 4) which was blocked with anti-CD4 (mean = 49.7 pglml), while levels of p24 viral protein in cultures of T cells only were somewhat higher than antiCD4 background levels (mean = 96.6 pglml). These data support our PCR results, which clearly showed increased HIV-l replication in DCscontaining cultures. While several groups have found that the APC function of DCs is ablated after exposure to HIV-I (Knight et al., 1993; Macatonia et al., 1989, 1991), we found that both allogeneic DCs (see Figure 4) and autologous DCs (Figure 3) could increase the levels of HIV-1 DNA, without being infected themselves. Although previous studies have shown that DCs have low levels of CD4 (Chehimi et al., 1993; Knight et al., 1990), in our studies DCs are not infected themselves. The fact that DC-dependent HIV-1 infection could be blocked by antiCD4, however, suggests that DCs promote the transfer of HIV-1 from infected to uninfected CD4+ T cells. To investigate this possibility more directly we added HIV-1 -pulsed T cells and DCs to uninfected B lymphocytes rather than CD4+ T lymphocytes. In this case, DCs did not increase HIV-1 DNA levels (data not shown). Thus, DC-dependent HIV-1 infection appears to be due to viral spread to uninfected CD4+ T cells rather than to enhanced replication in the CD4+ T cells initially infected. Anti-CD40 and Anti-CD28 Promote DC-Dependent HIV-1 Infection in CD4+ T Ceils To investigate the involvement of costimulatory molecules on DC-dependent infection, we treated DC-T cell cultures with MAbs specific for DC or T cell surface molecules. CD4+ T cells and DCs treated with anti-CD40 (10 pglml) showed 2.5 to 5-fold increases in HIV-1 DNA compared with CD4+ T cells plus DCs only (Figure 3A, lane 5; see Figure 5, lane 5), while T cells alone incubated with antiCD40 displayed very low levels of viral DNA (Figure 3A, lane 2; see Figure 5, lane 2). Although both activated monocytes and B cells can express CD40, anti-CD40 had no detectable effect on the low levels of viral DNA induced by monocytes (Figure 38, lane S), or B cells (Figure 3C, lane 5), indicating that the effect of anti-CD40 on HIV-1 DNA levels was due to its stimulatory action on DCs. In kinetic experiments, we tested the effect of anti-CD40 on CD4+ T cells, either alone or with 5 x lo4 autologous or allogeneic DCs for l-4 days of culture. Anti-CD40 induced increases in allogeneic DC-dependent HIV-1 infection by day 3, but only by day 4 when autologous DCs were used (data not shown). These results show that CD40 is functionally active on DCs (see also Figure 6), and suggest that activated T cells expressing CD40L may contribute to DC-dependent HIV transmission. To investigate the involvement of CD28 on DC-dependent HIV infection, we tested the effect of adding antiCD28(10 pg/ml) toCD4+Tcellscultured aloneor incombination with autologous DCs. CD4+ T cells cultured with

DCs had 4.8- to 5-fold increases in HIV-1 when treated with CD28 MAb (Figure 3D, lane 4), but T cells alone with anti-CD28 also showed 1.6- to 4.8-fold increases (Figure 3D, lane 2). These results agree with in vitro studies that anti-CD28 up-regulates HIV expression in infected T cells, (Van Lier et al., 1989). Unlike the increases in HIV-1 DNA levels induced by antiCD40, increases induced by antiCD28were not DC-dependent, suggesting that direct stimulation of T cells through CD28 bypasses the need for accessory molecules expressed on DCs. DGDependent HIV-1 DNA Expression Is Blocked by Anti-CD80 or CTLA4 lmmunoglobulin To investigate the involvement of other activation and adhesion molecules in DC-CD4+ T lymphocyte interactions during HIV-1 infection, we examined HIV-1 DNA synthesis when MAb to CD80, CD58, CD18, or MHC class II molecules (DR and DQ) were added separately to HIV-l-infected DC-CD4+ cell cultures. Soluble CTLA4 immunoglobulin, previously reported to block monocyte-dependent increases in HIV-1 (Deigel et al., 1993) (Figures 4IA and 4B), was also tested to inhibit CD28-CD80 interactions induced by allogeneic DCs. CD4+ T cells incubated with DCs displayed increased levels of viral DNA (Figure 4A, lane 5; Figure 4B, lane 4) which was blocked by anti-CD80 (Figure 4A, lane 6) or CTLA4 immunoglobulin (15 rkglml) (Figure 4A, lane 7). Both anti-DR (Figure 4B, lane 511or anti-DQ (Figure 4B, lane 6), but not anti-CD58 (Figure 4A, lane 8) or anti-CD18 (data not shown) blocked infection. Treatment of T cells alone with anti-CD80 (Figure 4A, lane 2), CTLA4 immunoglobulin (Figure 4A, lane 3) anti-DR (Figure 4B, lane 2), and DQ (Figure 4B, lane 3) ha.d no effect on levels of HIV-1 DNA, while anti-CD58 slightly increased these levels (Figure 4A, lane 4). Since MAb lo HLA-DR and HLA-DQ completely blocked the MLR stimulated by DCs, and since CTLA4 immunoglobulin and MAb to CD80 blocked DC-dependent T cell proliferation (Thomas et al., 1993a), our results are consistent with a model that DCdependent HIV-1 infection depends on T cell activation. These results are also consistent with a report showing that both T cell proliferation and IL-2 mRNA accumulation in HIV-l-infected T cell lines required costimulation via CD80 (Haffar et al., 1993). The inhibition of HIV-1 expression by anti-CD4 or MAb to DR and DQ may alct by either blocking viral spread or by inhibiting the activation of CD4+ T lymphocytes by DCs. Although our results with antiCD80 implicateCD80 in the regulation of HIV-1 expression in T cells, they do not rule out a possible rolle of other ligands for CD28 or CTLA-4 such as B70/87-2 (Azuma et al., 1993; Freeman et al., 1993), the activity of which may also have been blocked by CTLA4 immunoglobulin in our experiments. CTLA4 lmmunoglobulin Blocks DC-Dependent Increase in HIV-1 DNA Levels by Anti-CD48 To investigate whether anti-CD40 induced increases in DC-dependent HIV-1 expression could be lblocked by CTLA4 immunoglobulin, we added MAb to CD40 to HIV-linfected cultures in the presence or absence of CTLA4 immunoglobulin. In this experiment, treatment of T cells

Immunity 320

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Infected CD4+ T cells were added to uninfected CD4+ T cells alone or in combination with DCs (5 x IO4 autologous DCs) (A, D), 8 cells (5 x 10’) (C), or monocytes (5 x 10“) (B), with or without the indicated MAb. (A) Lanes 1-5, CD4+ T cells exposed to HIV-l, washed, recultured with uninfected CD4’ T cells and anti-CD4 (lane l), anti-CD40 (iane 2). Lane 3, CDQ T cells with DCs added. Lane 4, CD4’ T ceils plus DCs treated with antiCD4; lane 5, CD4+ T cells plus DCs treated with antiCD40. Lanes 6-9, controls. Lane 6, CD4+ T cells pulsed for 30 min with HIV-l and harvested immediately: lane 7, CD4+ T cells plus HIV-1 for 4 days; lane 6, CD4’ T cells pulsed for 30 min with HI HIV-1 and harvested immediately; lane 9, CD4+ T ceils plus HI HIV-1 for 4 days. Lanes IO-15 quantitate the amount of DNA equivalent to different numbers of cells. Lanes 16-20 quantitate the number of viral HIV-l copies represented by band intensity.

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(A) Lane l-8, CD4+ T cells incubated with HIV-1 for 30 min. washed, and then cultured 4 days with 4 x IO6 CD4+ T cells alone (lame 1). Lanes 2-4, CD4+ T cells pretreated with anti-CD80 (lane 2) CTLA4 immunoglobulin (lane 3) anti-CD58 (lane 4). Lanes 5-8, CD4+ T cells with 5 x IO4 allogeneic DCs. Lane 5, T cells and DCs alone. Lane 6, T plus DCs with antiCD80. Lane 7, T plus DCs with CTLA4 immunoglobulin. Lane 8, T plus DCs with anti-CD58. Lanes 9-16, controls. Lane 9, T cells without virus; lane 10, CD4+ T cells pulsed with HIV-1 (30 min) and harvested immediately. Lane 11, CD4+ T cells incubated with HIV-1 for 4 days. Lane 12, CD4+ T cells pulsed with HI HIV-1 (30 min) and harvested immediately. Lane 13, CD4+ T cells incubated with HI HIV-l for 4 days. Lanes 14-16 quantitate the number of viral HIV-l copies represented by band intensity. (B) Lanes l-6, CD4+ T cells incubated with HIV-l (30 min), washed, and added to 4 x lo6 CD4+ T cells for 4 days alone (lane 1). Lane 2-3, CD4’ T cells pretreated with anti-DR (lane 2), anti-DQ (lane 3). Lanes 4-6, CD4+ T cells plus DCs. Lane 4, T plus DCs alone. Lane 5, T plus DCs with anti-DR. Lane 6, T plus DCs with anti-DC Lanes 7-13, controls. Lane 7, T cells without virus. Lane 8, CD4+ T cells pulsed with HIV-1 (30 min) and harvested immediately. Lane 9, CD4+ T cells plus HIV-1 for 4 days. Lane 10, CD4+ T cells plus HI HIV-l for 4 days. Lanes 11-13, quantitate the number of viral HIV-1 copies represented by band intensity.

alone with anti-CD40 or CTLA4 immunoglobulin had no detectable effect on HIV-1 DNA levels (Figure 5, lanes 2 and 3). As shown previously, T cells cultured with DCs showed increased levels of viral DNA (Figure 5, lane 4), which could be augmented by anti-CD40 (Figure 5, lane 5) or blocked by CTLA4 immunoglobulin (Figure 5, lane 6). CTLA4 immunoglobulin also blocked augmentation of HIV-l levels induced by anti-CD40(Figure 5, lane 7). Thus, a CD80-CD28 (CTLA4) interaction appears to be required for anti-CD40-induced increases in HIV-l, perhaps by increasing levels of CD80 or other CD28 ligands. Activated CD4+ T cells can up-regulate the expression of CD80 on 6 cells (Ranheim and Kipps, 1993) and possibly on DCs;

CD80 in turn can signal TcellsviaCD28 (Clark and Ledbetter, 1994; Linsley and Ledbetter, 1993). CD28 costimulation on activated T cells enhances both IL-2 and IL-4 production and expression of CD40 ligand on activated Tcells (Clark and Ledbetter, 1994). This reciprocal dialogue between the CD40L-CD40 and CD80-CD28 cell--cell interaction pairs may be essential to promote HIV-1 expression and spread. MAb to CD40 Up-Regulate the Expression of CD80 and Other CTLA4 Ligands on DCs To test whether anti-CD40 induces increases in DCdependent HIV-1 replication via the regulation of the CD80

(B) Lanes l-3, controls. Lane 1, T cells without HIV-l; lane 2, T cells incubated with HI HIV-1 for 4 days; lane 3, CD4’ T cells incubated with HIV-1 for 4 days. Lane 4, CD4+ T cells exposed for 30 min to HIV-1 and harvested immediately. Lane 5, CD4+ T cells with monocytes for 4 days culture. Lane 6, CD4+ T cells plus monocytes treated with antiCD40. Lane 7, CD4+ T cells plus antiCD40. (C) Lanes 1-3, CD4’ T cells exposed to HIV-I, washed, recultured with uninfected CD4+ T cells alone (lane I), anti-CD40 (lane 2). anti-CD4 (lane 3). Lane 4, CD4+ T cells with B cells added; lane 5, CD4+ T cells plus B cells treated with anti-CD40; lane 6, CD4+ T cells plus B cells treated with anti-CD4; lanes 7-9, controls. Lane 7, CD4+ T cells pulsed for 30 min with HIV-l and harvested immediately; lane 8, CD4’ T cells plus HIV-1 for 4 days: lane 9, CD4+ T cells plus HI HIV-1 for 4 days. Lanes 10-15 quantitate the amount of DNA equivalent to different numbers of cells. Lanes 16-20 quantitate the number of viral HIV-l copies representedby band intensity. (D) Lanes 1-4, CD4+ T cells exposed to HIV-l for 30 min, washed, and then cultured 4 days with uninfected 4 x lOa CD4+ T cells alone (lane l), or treated with anti-CD28 (lane 2); lane 3, CD4+ T cells plus DCs; lane 4, CD4+ T cells plus DCs with anti-CDPB. Lanes 5-l 1, conbrols. Lane 5, T cells without virus: lane 6, CD4+ T cells pulsed with HIV-l for 30 min and harvested immediately; lane 7, CD4+ T cells incubated with HIV-1 for 4 days; lane 8, CD4’ T cells incubated with HI HIV-1 for 4 days. Lanes 9-l 1 quantitate the number of viral HIV-1 copies represented by band intensity.

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Lanes 7-7, CD4+ T cells plus HIV-l (30 min), washed, and then added to 4 x IO6 CD4+ T cells for 4 days alone (lane 1). Lanes 2-3, CD4‘ T cells pretreated with anti-CD40 (lane 2) CTLA4 immunoglobulin (lane 3). Lanes 4-7, CD4+ T cells with 5 x 10” autologous DCs. Lane 4, T plus DCs alone. Lane 5, T plus DCs with antiCD40. Lane 6, T plus DCs with CTLA4 immunoglobulin. Lane 7, T plus DCs with antiCD40 and CTLA4 immunoglobulin. Lanes 3-13, controls. Lane 8, T cells without virus. Lane 9, CD4’ T cells pulsed with HIV-1 (30 min) and harvested immediately. Lane 10, CD4+ T cells plus H-l HIV-1 (4 days). Lanes ! l-13, quantitate the number of viral HIV-l copies represented by band intensity.

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family, the expression of CD80- and CTLA4-binding proteins on DCs was analyzed by cytofluorography (see Experimental Procedures). DCs expressed low levels of CD80 and other CTLA4 ligands immediately after isolation (Figure 6). After treatment with an isotype control MAb, DCs expressed the same low levels of CD80 throughout 4 days of culture. In contrast, DCs treated with anti-CD40 had increased expression of CD80 and CTLA4 ligands detectable within day 1 after treatment, peaking by day 2, and decreasing byday4(Figure 6). CTLA4 immunoglobulin binding was significantly greater than CD80 MAb, suggesting, as has been reported for 6 cells (Azuma et al., 1993), that anti-CD40 increases expression of l370/87-2 on DCs. Under optimal culture conditions, treatment of DCs by anti-CD40 slightly augmented [3H]thymidine uptake of T cells in either allogeneic or autologous MLR, but this increase was not statistically significant (data not shown). Given that CD4+ T cells must be activated to replicate immunodeficiencyviruses (see Polacino et al., 1993), it is most likely that CD40 signaling of DCs up-regulates HIV-1 expression by promoting more efficient or prolonged activation of T cells via CD80 and B70/87-2. The fact that CTLA4 immunoglobulin blocks DC-dependent HIV-1 viral spread in vitro even in the presence of a DC stimulator (anti-CD40) suggests CTLA4 immunoglobulin may have a therapeutic potential to reduce viral burden in AIDS. Together with P. Linsley and coworkers, we are currently testing this possibility in a simian immunodeficiency virus model in macaques.

Experimental

Procedures

Antibodies and Fusion Proteins Purified MAb to CD19 (HD37), CD40 (G28-5), CD45RA(3AC5), fluorescein- and phycoerythrin-conjugated MAb to CD3 (G19-4), CD14 (LeuM3), CD16 (Leu-lla), CD20 (lF5), streptoavidin R-phycoerythrin and fluorescein conjugates (Tago, Incorporated) were used in the purification of DCs, B cells, and monocytes. A MAb to CD8 (610-l) was used in the purification of CD4+ T lymphocytes. MAb to CD40 (G28-5) CD4 (OKT4a), CD80 (L310), CD58 (TS2/9), CD18 (60.3) CD28 (9.3). anticlass II DR (HE1 Oa), anti-class II DQ (4AA7), and fusion proteins CTLA4 immunoglobulin (provided by Dr. P. Linsley et al., 1991) were used in investigating of DC-dependent HIV-1 infection. Dendritic Gelis An improved enrichment procedure for human blood DCs was used (Freudenthal and Steinman, 1990) with some minor modifications. In brief, mononuclear cells were isolated on Ficoll-Hypaque gradients and E rosette- (Er-) cells were cultured for 18-24 hr. Monocytes were removed by two rounds of adherence to plastic, then nonadherent cells were “panned” using MAb to CD19 (10 pglml) to remove B cells. Discontinuous Percoll gradients were used to isolate the low density (< 50.5%) faction. A combination of MAb to markers that selectively identify other cell types (CD3, CD14, CDt6, and CD20 for T cells, monocytes, natural killers, and B cells, respectively) was used toselect negatively the large (high forward scatter) marker-negative DC subset with a fluorescence-activated cell sorter (FACStar Plus, Becton Dickinson lmmunocytometry Systems, San Jose, California). The resulting population of DCs was examined for purity with a FACScan (Eecton Dickinson lmmunocytometry Systems, Sa Jose, California) using fluorescein-conjugated antibodies to CD3, CD16, CD19, CD20, and CD14 with single or two color analysis, or using biotin-anti-CD40 followed by a streptoavidin R-phycoerythrin conjugate (red) versus a cocktail of fluorescein-conjugated antibodies to CD3, CD16, CD20, and CD14 (green) (Figure la). To investigate the expression of CD80 or other

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MLR Stimulators (APCs) for MLR were derived from the separation protocol shown above. The APCs were treated with mitomycin C (100 uglml) or were irradiated (3000 rads i37Cs) and added in graded doses to lo5 allogeneic or syngeneic Er+ cells. Proliferation was measured by the uptake of PH]thymidine (1 pCi per well added for 18 hr on the fifth day). Responses are reported as mean cpm of triplicates, omitting the standard deviations, which were
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Monocytes and Lymphocytes In some experiments, a CD14 MAb (Leu-M3) was used to isolate large CD14+ monocytes with the FACS. The purity of the resulting population was 96%-980/o. Fluorescein-conjugated CD20 MAb was used to isolate B cells with the FACS. The purity of the resulting population was usually 97%-98%. To isolate CD4+ T cells, Er+ cells were further depleted by panning twice on plastic dishes coated by anti-CD8 (10 kg/ml). The purity of CD4+ cells was usually 940/o-96%. In some exeriments, we stimulated CD4+ T cells with 1 nglml of the staphylococcal enterotoxin B (Toxin Technology). B lymphocytes were isolated during the purification of DCs. After panning Er population with a CD19 MAb, adherent CDlS+ cells were removed and put in culture with HIV-I. The purity of B cells was >90%.

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DCs were gated based on large forward scatter and negative staining by a panel of PE-conjugated cell-specific MAbs to B and T lymphocytes, monocytes, and natural killers. This excluded nondendritic cell leukocytes from the analysis at time 0, day 2, and day 4 after activation. DCs were assessed for expression of CD80 family members, using biotinylated antiCD80, CTLA4 immunoglobulin, or isotype-matched control antibodies followed by a streptoavidin-fluorescein conjugates. Isotype-matched MAb (1) to anti-CD80 (line); Human IgG control (2) for CTLA4 immunoglobulin (dotted line); anti-CD80 (3) (short broken line); CTLA4 immunoglobulin (4) (long broken line).

CTLA4 ligands on DCs, low density fractions were used after panning on anti-CD45RA-coated dishes. This DC-enriched population usually was 40%-60% pure. The cells were analyzed by FACS as described (Young et al., 1992) with some modifications. Part of the cells was analyzed by gating on the cell population with large forward scatter that did not stain with a panel of phycoerythin-congugated cell-specific MAbs to CD3, CD19, CD20, CD14, CD16 for T cells, B cells, monocytes, and natural killers. Having excluded nondendritic cell PBMC from the analysis gate, DCs were then analyzed for positive counterstaining by using biotin-anti-CD80, CTLA4 immunoglobulin, or isotypematched MAb followed by a streptoavidin fluorescein conjugates (Tago, Incorporated).

HIV-1 Infection and Detection HIV-lLaI (Barre-Sinoussi et al., 1983; Wain-Hobson et al., 1991) was prepared as described previously (Agy et al., 1990) and treated with DNAase just prior to infection to minimize viral DNA contamination of theinfectiousvirusstock(Polacinoetal., 1993).Thecellswereinfected at a multiplicity of infection approximately0.01 tissue culture infectious dose. In some experiments, purified DCs, B cells, and stimulated CD4+ T cells (staphylococcal enterotoxin B [1 nglml]) were used as mitogenic stimulant), PBMC were either mock infected with HI virus or infected with HIV-I LRIand cultured for 16 hr. Cells were washed, free of culture supernatant, extracts were prepared in lysis buffer 0.45% NP40, 0.45 % Tween 20, 50 mM Tris (pH 8.3) and 125 mM KCI. ‘The extracts were proteinase K (600 pglml) digested for 2 hr at 37OC followed by 10 min in a boiling water bath. Of the sample mix, 20 pl (approximately lo5 cells) were used for PCR using gag-specific primers (SK 38, 39) (Ou et al., 1988). For other experiments, purified CD4+ T cells (4 x 109 were either mock or virus infected. Following a 30 min attachment, the cells were washed free of unattached virus and resuspended in complete culture medium containing IO U/ml of IL-2 (Boehringer Mannheim Biochemicals). Immediately following washing, 2 x IO4 infected CD4+ T cells were added to uninfected cultures containing 4 x IO6 CD4+ T cells (in some experiments we used 4 x 10” uninfected B cells) alone or with APCs and cultured under standard conditions (37°C with 5%-10% Con). Lysates were prepared daily for 4 days. In these experiments, PCR detection of HIV-l-specific nucleic acid sequences was performed on 20 ng of total cellular DNA (4 x IO3 cells) and analyzed by electrophoresis as described previously (Poslacino et al., 1993). For all PCR we used HIV-l-specific primers to gag region SK 38 and SK 39, yielding a 115 bp fragment. Products of the f3-globin gene were simultaneously analyzed with primers PC04 and GH20, yielding a 288 bp fragment, to normalize the amount of DNA (PerkinsElmer Cetus). For some experiments, we performed densitometric analysis on the bands revealed by autoradiography. HIV protein levels were determined in culture supernatants using a whole viral antigen capture enzyme-linked immunosorbent assay(Abott Laboratories) following the instructions of the manufacturer.

Acknowledgments We wish to thank M. Toumaforassistance in cell sorting. The technical assistance of K. Milligan and A. Liang is gratefully acknowledged. We thank Drs. D. Buck, J. Hansen, J. Ledbetter, T. Pazzutto, and T. Springer for monoclonal antibodies and Dr. P. Linsley for CTLA4 immunoglobulin fusion protein. This work was supported by National Institutes of Health grants RR00166 (Regional Primate Research Center) and GM 37905 (E. A. C.).

Immunity 324

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January

12, 1994; revised

May 13, 1994.

dendritic cells with HIVI: virus load regulates stimulation sion of T-cell activity. Res. Viral. 744, 75-80.

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