Long-Term Productive Human Immunodeficiency Virus
http://www.100md.com
病菌学杂志 2005年第1期
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Department of Medicine, Harvard Medical School, Boston, Massachusetts
ABSTRACT
Myeloid, CD1a-sorted dendritic cells (MDC) productively replicated human immunodeficiency virus strains encoding envelope genes of either primary X4R5 or R5 strains for up to 45 days. Cell-free supernatant collected from long-term infected MDC, which had been exposed to an X4R5 virus 45 days earlier, was still infectious when placed over activated T cells. These data imply that DC can act as a persistent reservoir of infectious virus.
TEXT
Dendritic cells (DC) play a crucial role in the immune defense against pathogens (4, 24). Two subsets, plasmacytoid DC and myeloid DC (MDC), were identified in humans based on their phenotypes (5). MDC are involved in uptake, processing, and presentation of foreign antigens. Immature MDC are disseminated throughout tissues, where they capture antigens, mature, and home to lymph nodes; DC then present the processed antigens to T cells (20).
A number of groups have examined the ability of MDC to interact with human immunodeficiency virus (HIV) (1, 2, 6, 17, 23, 30, 33). Recent studies demonstrated that HIV replication depends on the stage of DC differentiation (3, 19, 21). DC express CD4 and the chemokine receptors CCR5 and CXCR4 (2, 13, 29, 34); the expression of the latter molecules varies with the DC maturation stage (3, 7), thus influencing the cells' susceptibility to productive HIV infection. Also, HIV can interact with DC via the C-type lectin DC-SIGN without undergoing replication; captured virus remains infectious for days and can be transmitted to T cells (12).
The aim of this study was to determine the period of time during which highly purified MDC can support replication of HIV to explore whether virus-exposed MDC could represent a potential long-lived reservoir of infectious HIV. For this purpose, we established long-term cultures of MDC that were differentiated from peripheral blood monocytes (Fig. 1). After 6 days of differentiation in complete RPMI 1640 medium supplemented with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (25, 26), we observed cells with a veiled appearance (Fig. 2A). These cells were purified by positive selection against CD1a (Fig. 2B to E). Staining of CD1a-sorted MDC with monoclonal antibodies (MAbs) against CD3, CD14, and CD19 did not show contamination with T cells, macrophages, and B cells (data not shown). After sorting, these MDC were DC-SIGN+ (Fig. 2F) and expressed markers typical for immature MDC; a minor fraction of these cells expressed CCR5 or CXCR4 (Table 1). CD1a-sorted MDC performed macropinocytosis efficiently; when the cells were matured with lipopolysaccharide (LPS), their ability to perform macropinocytosis decreased dramatically (Fig. 2G). Thus, our DC were highly pure and exhibited properties of immature MDC.
We exposed the CD1a-sorted MDC to HIV-GFP, an X4R5 virus containing the envelope gene of the dual-tropic, primary HIV isolate 89.6 (9) and the gene for green fluorescent protein (GFP). Like the parental HIV89.6, HIV-GFP was able to use both CXCR4 and CCR5 for virus entry, while the reporter gene GFP placed in lieu of nef allowed detection of infection at the single-cell level. Because we wanted to show that DC could represent a reservoir of productive HIV replication during chronic infection, we chose this dual-tropic HIV strain as a representative of a primary HIV isolate that can be found during chronic infection. Throughout disease progression, some HIV strains expand their usage of coreceptors for viral entry, shifting from exclusive use of CCR5 to use of CXCR4; this shift has been described for approximately half of the HIV clade B-infected individuals with progressive disease (10, 27, 32).
Our purified MDC were highly positive for DC-SIGN and expressed both CCR5 and CXCR4 (Fig. 2F; Table 1). As expected, these MDC were productively infected by the dual-tropic X4R5 HIV-GFP (Fig. 3). Virus-exposed MDC were cultured for 45 days, and throughout this time period, GFP-expressing and hence productively infected MDC were detected (Fig. 3A and B). In general, between 1.5 to 4.5% of the virus-exposed MDC were GFP+ and thus HIV+. Supernatants from infected DC collected at various time points contained p24 Gag that peaked on day 10 and was present in culture supernatants during the next 35 days (Fig. 3C). MDC on day 30 still expressed DC-SIGN at high levels (Table 1), and we were able to colocalize GFP and DC-SIGN, indicating productive HIV infection of DC at the single-cell level (Fig. 3D). On day 45 of culture, the viability of virus-exposed MDC and that of unexposed controls was 63 and 70%, respectively (Table 2). Both cultures were CD14 negative; no surface CD14 expression was found on GFP-expressing, HIV-infected cells (data not shown). The absolute number of virus-exposed and nonexposed control cells decreased from 3 x 107 on day one to 1.9 x 107 and 2.1 x 107 on day 45 after viral exposure, respectively. These data indicate that MDC can be maintained in culture for extended periods of time with the majority of the cells remaining viable. Furthermore, productive HIV replication by a small fraction of GFP+ cells did not appreciably influence the long-term viability of the entire culture.
The use of dual-tropic HIV-GFP allowed us to examine whether long-term productively infected MDC could transfer viral progeny to T cells. On day 45 after virus exposure, MDC were separated by flow cytometry into GFP+ and GFP– fractions (Fig. 1 and Fig. 4B, C, and D); approximately 4.5% of the MDC were GFP+ and thus productively infected. Real-time DNA-PCR with primers specific for HIV pol was used to demonstrate the presence of proviral DNA. A strong signal was detected in GFP+ cells (Fig. 4E), indicating that HIV-GFP entered MDC, proceeded through the early steps of the viral life cycle, and completed reverse transcription.
Next, the sorted GFP+ cells were plated in the upper wells of transwell tissue culture units (103 cells/well), while the lower wells contained CEMx174 cells (5 x 104 cells). As negative controls, nonexposed MDC were placed into the upper wells in parallel plates. GFP+ MDC released HIV that crossed the semipermeable membrane of the transwell unit and infected CEMx174 cells; release of p24 Gag into the culture supernatant increased as a function of time (Fig. 5A). In parallel, we also examined the MDC fraction that was GFP negative 45 days after initial exposure to HIV-GFP. Only a low level of viral replication was detected in the transwell system involving CEMx174 cells in the bottom wells (Fig. 5A), possibly due to the presence of a few MDC, in which infectious HIV-GFP had initiated the replication cycle without yet expressing GFP at sufficient levels at the time of sorting.
We then investigated whether sorted GFP+ MDC could transmit HIV to primary autologous T cells. GFP+ MDC were placed into the upper wells of transwell units, while the lower wells contained either autologous T cells alone or T cells cocultured with autologous, freshly differentiated, noninfected MDC at a ratio of 10:1. In the latter culture, p24 Gag increased as a function of time (Fig. 5B); in the absence of MDC, virus replicated at a substantially lower level (120 ng/ml versus 27 ng/ml of p24 on day 15 of incubation). This indicated that GFP+ MDC in the upper wells shed virus that was propagated efficiently by the T cells activated by autologous DC but not by T cells cultured alone. Together, our data clearly demonstrate that 45 days after virus exposure, MDC harbored infectious virus that could be transmitted in the absence of cell-cell contact.
To investigate whether MDC can support long-term productive infection of a primary HIV isolate, we exposed CD1a-sorted MDC to HIV1084i, an R5 primary HIV clade C strain from Africa (14). This virus, which had been isolated from a 4-month-old infant infected either intrapartum or by breast feeding, was molecularly cloned and tested for its tropism (14). We maintained HIV-exposed and nonexposed MDC cultures for 45 days in the presence or absence of azidothymidine (AZT) (Fig. 5C). Clearly, MDC supported the replication of this strain, indicating permissiveness to both X4R5 (Fig. 3C) and R5 viruses.
In summary, CD1a+ sorted MDC supported HIV replication for 45 days. Progeny virus released by GFP+ MDC on day 45 was fully infectious and readily replicated in the CEMx174 cell line and in primary T cells cocultured with uninfected autologous MDC. T cells upon contact with noninfected MDC undergo activation, which leads to the upregulation of HLA-DR, CD25, and CD69 (31). According to earlier data, activation of T cells is one of prerequisites for productive HIV replication (8, 22, 28). We found that even a low amount of HIV released by the long-term infected MDC was sufficient to rapidly spread infection in the MDC-T-cell microenvironment.
We demonstrated here for the first time that CD1a-sorted, DC-SIGN+, HIV-exposed MDC survive and release infectious virus during a protracted period of time. It would be interesting to compare the life span of our long-term in vitro HIV-infected DC with the life span of human DC in vivo. Few data are available about the life span of DC. Earlier studies reported that Langerhans cells represent a long-lived cell population (15, 16, 18). For instance, Langerhans cells were identified throughout 9 weeks in human skin grafts, which were transplanted to BALB/c nude mice (18). MDC are motile cells circulating in peripheral blood, residing in mucosa, and easily migrating across vascular endothelium (5, 11). Our data imply that MDC could form a long-lived, motile HIV reservoir with an important role in disseminating infectious virus through peripheral blood and in lymphoid and nonlymphoid tissues. Both productive infection and DC-SIGN-mediated virus capture could contribute to HIV transmission in vivo.
ACKNOWLEDGMENTS
We thank H. G?ttlinger, Worcester, Mass., for providing the dual-tropic HIV-GFP strain, P. Autissier for sorting of HIV-infected DC, S. Sharp for help in preparing the manuscript, and D. S. Popov for assistance with the figures.
This work was supported by NIH grants RO1 AI43839, RO1 DE12937, and RO1 DE016013 to R.M.R. and by Center for AIDS Research core grant IP3028691 awarded to the Dana-Farber Cancer Institute as support for AIDS research efforts by the institute.
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ABSTRACT
Myeloid, CD1a-sorted dendritic cells (MDC) productively replicated human immunodeficiency virus strains encoding envelope genes of either primary X4R5 or R5 strains for up to 45 days. Cell-free supernatant collected from long-term infected MDC, which had been exposed to an X4R5 virus 45 days earlier, was still infectious when placed over activated T cells. These data imply that DC can act as a persistent reservoir of infectious virus.
TEXT
Dendritic cells (DC) play a crucial role in the immune defense against pathogens (4, 24). Two subsets, plasmacytoid DC and myeloid DC (MDC), were identified in humans based on their phenotypes (5). MDC are involved in uptake, processing, and presentation of foreign antigens. Immature MDC are disseminated throughout tissues, where they capture antigens, mature, and home to lymph nodes; DC then present the processed antigens to T cells (20).
A number of groups have examined the ability of MDC to interact with human immunodeficiency virus (HIV) (1, 2, 6, 17, 23, 30, 33). Recent studies demonstrated that HIV replication depends on the stage of DC differentiation (3, 19, 21). DC express CD4 and the chemokine receptors CCR5 and CXCR4 (2, 13, 29, 34); the expression of the latter molecules varies with the DC maturation stage (3, 7), thus influencing the cells' susceptibility to productive HIV infection. Also, HIV can interact with DC via the C-type lectin DC-SIGN without undergoing replication; captured virus remains infectious for days and can be transmitted to T cells (12).
The aim of this study was to determine the period of time during which highly purified MDC can support replication of HIV to explore whether virus-exposed MDC could represent a potential long-lived reservoir of infectious HIV. For this purpose, we established long-term cultures of MDC that were differentiated from peripheral blood monocytes (Fig. 1). After 6 days of differentiation in complete RPMI 1640 medium supplemented with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (25, 26), we observed cells with a veiled appearance (Fig. 2A). These cells were purified by positive selection against CD1a (Fig. 2B to E). Staining of CD1a-sorted MDC with monoclonal antibodies (MAbs) against CD3, CD14, and CD19 did not show contamination with T cells, macrophages, and B cells (data not shown). After sorting, these MDC were DC-SIGN+ (Fig. 2F) and expressed markers typical for immature MDC; a minor fraction of these cells expressed CCR5 or CXCR4 (Table 1). CD1a-sorted MDC performed macropinocytosis efficiently; when the cells were matured with lipopolysaccharide (LPS), their ability to perform macropinocytosis decreased dramatically (Fig. 2G). Thus, our DC were highly pure and exhibited properties of immature MDC.
We exposed the CD1a-sorted MDC to HIV-GFP, an X4R5 virus containing the envelope gene of the dual-tropic, primary HIV isolate 89.6 (9) and the gene for green fluorescent protein (GFP). Like the parental HIV89.6, HIV-GFP was able to use both CXCR4 and CCR5 for virus entry, while the reporter gene GFP placed in lieu of nef allowed detection of infection at the single-cell level. Because we wanted to show that DC could represent a reservoir of productive HIV replication during chronic infection, we chose this dual-tropic HIV strain as a representative of a primary HIV isolate that can be found during chronic infection. Throughout disease progression, some HIV strains expand their usage of coreceptors for viral entry, shifting from exclusive use of CCR5 to use of CXCR4; this shift has been described for approximately half of the HIV clade B-infected individuals with progressive disease (10, 27, 32).
Our purified MDC were highly positive for DC-SIGN and expressed both CCR5 and CXCR4 (Fig. 2F; Table 1). As expected, these MDC were productively infected by the dual-tropic X4R5 HIV-GFP (Fig. 3). Virus-exposed MDC were cultured for 45 days, and throughout this time period, GFP-expressing and hence productively infected MDC were detected (Fig. 3A and B). In general, between 1.5 to 4.5% of the virus-exposed MDC were GFP+ and thus HIV+. Supernatants from infected DC collected at various time points contained p24 Gag that peaked on day 10 and was present in culture supernatants during the next 35 days (Fig. 3C). MDC on day 30 still expressed DC-SIGN at high levels (Table 1), and we were able to colocalize GFP and DC-SIGN, indicating productive HIV infection of DC at the single-cell level (Fig. 3D). On day 45 of culture, the viability of virus-exposed MDC and that of unexposed controls was 63 and 70%, respectively (Table 2). Both cultures were CD14 negative; no surface CD14 expression was found on GFP-expressing, HIV-infected cells (data not shown). The absolute number of virus-exposed and nonexposed control cells decreased from 3 x 107 on day one to 1.9 x 107 and 2.1 x 107 on day 45 after viral exposure, respectively. These data indicate that MDC can be maintained in culture for extended periods of time with the majority of the cells remaining viable. Furthermore, productive HIV replication by a small fraction of GFP+ cells did not appreciably influence the long-term viability of the entire culture.
The use of dual-tropic HIV-GFP allowed us to examine whether long-term productively infected MDC could transfer viral progeny to T cells. On day 45 after virus exposure, MDC were separated by flow cytometry into GFP+ and GFP– fractions (Fig. 1 and Fig. 4B, C, and D); approximately 4.5% of the MDC were GFP+ and thus productively infected. Real-time DNA-PCR with primers specific for HIV pol was used to demonstrate the presence of proviral DNA. A strong signal was detected in GFP+ cells (Fig. 4E), indicating that HIV-GFP entered MDC, proceeded through the early steps of the viral life cycle, and completed reverse transcription.
Next, the sorted GFP+ cells were plated in the upper wells of transwell tissue culture units (103 cells/well), while the lower wells contained CEMx174 cells (5 x 104 cells). As negative controls, nonexposed MDC were placed into the upper wells in parallel plates. GFP+ MDC released HIV that crossed the semipermeable membrane of the transwell unit and infected CEMx174 cells; release of p24 Gag into the culture supernatant increased as a function of time (Fig. 5A). In parallel, we also examined the MDC fraction that was GFP negative 45 days after initial exposure to HIV-GFP. Only a low level of viral replication was detected in the transwell system involving CEMx174 cells in the bottom wells (Fig. 5A), possibly due to the presence of a few MDC, in which infectious HIV-GFP had initiated the replication cycle without yet expressing GFP at sufficient levels at the time of sorting.
We then investigated whether sorted GFP+ MDC could transmit HIV to primary autologous T cells. GFP+ MDC were placed into the upper wells of transwell units, while the lower wells contained either autologous T cells alone or T cells cocultured with autologous, freshly differentiated, noninfected MDC at a ratio of 10:1. In the latter culture, p24 Gag increased as a function of time (Fig. 5B); in the absence of MDC, virus replicated at a substantially lower level (120 ng/ml versus 27 ng/ml of p24 on day 15 of incubation). This indicated that GFP+ MDC in the upper wells shed virus that was propagated efficiently by the T cells activated by autologous DC but not by T cells cultured alone. Together, our data clearly demonstrate that 45 days after virus exposure, MDC harbored infectious virus that could be transmitted in the absence of cell-cell contact.
To investigate whether MDC can support long-term productive infection of a primary HIV isolate, we exposed CD1a-sorted MDC to HIV1084i, an R5 primary HIV clade C strain from Africa (14). This virus, which had been isolated from a 4-month-old infant infected either intrapartum or by breast feeding, was molecularly cloned and tested for its tropism (14). We maintained HIV-exposed and nonexposed MDC cultures for 45 days in the presence or absence of azidothymidine (AZT) (Fig. 5C). Clearly, MDC supported the replication of this strain, indicating permissiveness to both X4R5 (Fig. 3C) and R5 viruses.
In summary, CD1a+ sorted MDC supported HIV replication for 45 days. Progeny virus released by GFP+ MDC on day 45 was fully infectious and readily replicated in the CEMx174 cell line and in primary T cells cocultured with uninfected autologous MDC. T cells upon contact with noninfected MDC undergo activation, which leads to the upregulation of HLA-DR, CD25, and CD69 (31). According to earlier data, activation of T cells is one of prerequisites for productive HIV replication (8, 22, 28). We found that even a low amount of HIV released by the long-term infected MDC was sufficient to rapidly spread infection in the MDC-T-cell microenvironment.
We demonstrated here for the first time that CD1a-sorted, DC-SIGN+, HIV-exposed MDC survive and release infectious virus during a protracted period of time. It would be interesting to compare the life span of our long-term in vitro HIV-infected DC with the life span of human DC in vivo. Few data are available about the life span of DC. Earlier studies reported that Langerhans cells represent a long-lived cell population (15, 16, 18). For instance, Langerhans cells were identified throughout 9 weeks in human skin grafts, which were transplanted to BALB/c nude mice (18). MDC are motile cells circulating in peripheral blood, residing in mucosa, and easily migrating across vascular endothelium (5, 11). Our data imply that MDC could form a long-lived, motile HIV reservoir with an important role in disseminating infectious virus through peripheral blood and in lymphoid and nonlymphoid tissues. Both productive infection and DC-SIGN-mediated virus capture could contribute to HIV transmission in vivo.
ACKNOWLEDGMENTS
We thank H. G?ttlinger, Worcester, Mass., for providing the dual-tropic HIV-GFP strain, P. Autissier for sorting of HIV-infected DC, S. Sharp for help in preparing the manuscript, and D. S. Popov for assistance with the figures.
This work was supported by NIH grants RO1 AI43839, RO1 DE12937, and RO1 DE016013 to R.M.R. and by Center for AIDS Research core grant IP3028691 awarded to the Dana-Farber Cancer Institute as support for AIDS research efforts by the institute.
REFERENCES
Ancuta, P., Y. Bakri, N. Chomont, H. Hocini, D. Gabuzda, and N. Haeffner-Cavaillon. 2001. Opposite effects of IL-10 on the ability of dendritic cells and macrophages to replicate primary CXCR4-dependent HIV-1 strains. J. Immunol. 166:4244-4253.
Ayehunie, S., E. A. Garcia-Zepeda, J. A. Hoxie, R. Horuk, T. S. Kupper, A. D. Luster, and R. M. Ruprecht. 1997. Human immunodeficiency virus-1 entry into purified blood dendritic cells through CC and CXC chemokine coreceptors. Blood 90:1379-1386.
Bakri, Y., C. Schiffer, V. Zennou, P. Charneau, E. Kahn, A. Benjouad, J. C. Gluckman, and B. Canque. 2001. The maturation of dendritic cells results in postintegration inhibition of HIV-1 replication. J. Immunol. 166:3780-3788.
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