extfieldsofstudy: Biology Medicine

text:
Yersinia virulence factor YopJ acts as a deubiquitinase to inhibit NF-κB activation

The bacterial pathogens of the genus Yersinia, the causative agents of plague, septicemia, and gastrointestinal syndromes, use a type III secretion system to inject virulence factors into host target cells. One virulence factor, YopJ, is essential for the death of infected macrophages and can block host proinflammatory responses by inhibiting both the nuclear factor κB (NF-κB) and mitogen-activated protein kinase pathways, which might be important for evasion of the host immune response and aid in establishing a systemic infection. Here, we show that YopJ is a promiscuous deubiquitinating enzyme that negatively regulates signaling by removing ubiquitin moieties from critical proteins, such as TRAF2, TRAF6, and IκBα. In contrast to the cylindromatosis tumor suppressor CYLD, which attenuates NF-κB signaling by selectively removing K63-linked polyubiquitin chains that activate IκB kinase, YopJ also cleaves K48-linked chains and thereby inhibits proteasomal degradation of IκBα. YopJ, but not a catalytically inactive YopJ mutant, promoted deubiquitination of cellular proteins and cleaved both K48- and K63-linked polyubiquitin. Moreover, an in vitro assay was established to demonstrate directly the deubiquitinating activity of purified YopJ.

Pathogenic Yersinia, Yersinia pseudotuberculosis , and Yersinia enterocolitica must express the cysteine protease YopJ to kill infected macrophages and establish a systemic infection in mice (1)(2)(3)(4). Other members of the family of cysteine proteases to which YopJ belongs include the adenovirus protease AVP, the proapoptotic protein AvrA of Salmonella typhimurium, and the Xanthomonas campestris pv. vesicatoria protein AvrBsT, which induces localized cell death in infected plant leaves (4). Mutation of the conserved catalytic cysteine in YopJ abolishes its ability to inhibit the proinflammatory mitogen-activated protein kinase (MAPK) and NF-B pathways that are activated within infected cells, and it has been suggested that YopJ proteolytic activity is required to remove the ubiquitin-like protein SUMO-1 from posttranslationally modified proteins (4). However, the proteins that must be de-sumoylated to shut down NF-B or MAPK signaling remain ill defined. In this study, we identify YopJ as a promiscuous deubiquitinating protease that negatively regulates the host cell response by cleaving ubiquitin moieties from critical proteins, such as TRAF2, TRAF6, and I B ␣ .

RESULTS AND DISCUSSION
To identify the YopJ substrates that are critical to NF-B signaling, we began by comparing the impact of YopJ on NF-B activation in response to a variety of different stimuli. HEK293T cells were cotransfected with YopJ, a NF-B-dependent reporter gene, and one of the following activators of NF-B transcription: TRAF6, which is essential for NF-B signaling when LPS engages Toll-like receptor 4; TRAF2 or TRAF5, which signal NF-B activation downstream of TNF receptor 1; NIK, which signals NF-B activation downstream of the lymphotoxin ␤ receptor and the BAFF/ BLyS receptor 3; IKK ␣ or IKK ␤ , components of the I B kinase (IKK) complex that is activated by diverse stimuli to phosphorylate I B proteins and thereby targets them for ubiquitinmediated degradation; and c-Rel, a member of the NF-B family of transcription factors ( Fig.  1 A and  C172A. These results indicate that YopJ can inhibit NF-B signaling in response to diverse stimuli and that inhibition is dependent on its protease activity. WT YopJ did not, however, block NF-B activation after c-Rel overexpression, indicating that YopJ does not have a direct effect on the transcriptional activity of NF-B (Fig. 1 A).
Given that SUMO-1-conjugated proteins have been reported as targets for YopJ (4), we next examined whether sumoylation of signaling components is a requirement for NF-B activation, such that removal of SUMO-1 by YopJ might lead to inhibition of the NF-B pathway. To test this hypothesis, we transfected 293T cells with siRNA against Ubc9, the E2 enzyme essential for protein sumoylation (8)(9), and determined whether TRAF2, TRAF6, or IKK ␤ still activated an NF-B-dependent reporter gene (Fig. 1 B). Significant knockdown of Ubc9 expression was confirmed by West-ern blotting ( Fig. 1 C), but there was no inhibition of TRAF2-, TRAF6-, or IKK ␤ -induced NF-B activity (Fig. 1  B). We verified that Ubc9 knockdown inhibited sumoylation by Western blotting of RanGAP1, a known target for sumoylation (10)(11). The slower migrating form of RanGAP1 conjugated to SUMO-1 was abolished in cells transfected with Ubc9 siRNA (Fig. 1 D). These data suggest that sumoylation may not be required for NF-B activation, at least in response to expression of TRAF2, TRAF6, or IKK ␤ , which brings into question the notion that YopJ inhibits NF-B signaling by functioning as a SUMO-1 isopeptidase.
Recent studies have identified a critical role for ubiquitination in NF-B signaling. For example, TRAF2 and TRAF6 undergo autoubiquitination and acquire polyubiquitin chains in which lysine 63 (K63) of one ubiquitin is linked to the COOH terminus of an adjacent ubiquitin. Modification of TRAF2 or TRAF6 in this manner is proposed to promote the assembly of a multiprotein complex that activates the IKK complex and hence, NF-B signaling (12)(13)(14). The cylindromatosis tumor suppressor protein CYLD recently was defined as a deubiquitinating protease that inhibits TRAF2-and TRAF6-induced NF-B activation by cleaving K63-linked ubiquitin chains (15)(16)(17). Therefore, we explored whether YopJ might also inhibit the NF-B pathway by targeting ubiquitinated proteins rather than proteins conjugated to SUMO-1. To facilitate the detection of ubiquitin and SUMO-1 conjugates, 293T cells expressing YopJ were also transfected with SUMO-1 or ubiquitin having NH 2 -terminal hemagglutinin (HA) epitope tags. Immunoblotting of whole cell lysates with HA antibodies revealed that cells expressing YopJ contained far fewer ubiquitinated proteins than cells not expressing YopJ (Fig. 2 A, left). By contrast, YopJ did not decrease total cellular sumoylated protein levels ( Fig. 2 A, right). The decrease in cellular ubiquitinated proteins mediated by YopJ was dependent on YopJ protease activity because the catalytic cysteine YopJ mutant C172A did not reduce ubiquitinated protein levels ( Fig. 2 A). Consistent with the work of others (10)(11)(12), basal levels of total cellular ubiquitinated proteins in 293T cells, but not sumoylated proteins, were elevated further by overexpression of TRAF2 or TRAF6 (Fig. 2 B and not depicted). WT YopJ, but not YopJ mutant C172A, prevented this increased level of ubiquitinated proteins, further supporting the possibility that YopJ inhibits NF-B signaling by acting as a deubiquitinating protease rather than a SUMO-1 isopeptidase.
To test whether ubiquitinated TRAF2 and TRAF6 are substrates for YopJ, TRAF2 or TRAF6 was overexpressed with HA ubiquitin and YopJ and then immunoprecipitated from denatured cell lysates and immunoblotted with HA antibody. YopJ, but not YopJ mutant C172A, abolished TRAF2 and TRAF6 autoubiquitination (Fig. 2 C). This inhibition of TRAF2 and TRAF6 autoubiquitination by YopJ correlated not only with inhibition of NF-B signaling ( Fig.  1 A), but also with impaired activation of extracellular-regulated kinases (ERKs) 1 and 2, Jun NH 2 -terminal kinases 1

BRIEF DEFINITIVE REPORT
and 2, and p38 MAPK (Fig. 2 D; references [18][19][20]. These results suggested that YopJ, like CYLD, prevents NF-B signaling by acting as a deubiquitinating protease. However, when we compared the activities of YopJ and CYLD in our assay systems (Fig. 3), differences between the two were apparent. Although YopJ inhibited expression of an NF-Bdependent reporter in response to overexpression of TRAF2, TRAF6, IKK ␣ , IKK ␤ , and NIK, CYLD was only effective at blocking NF-B signaling by TRAF2 and TRAF6 (Fig. 3, A and B). We also tested whether the lysine residue in ubiquitin that is used to assemble polyubiquitin chains impacts deubiquitination by YopJ and CYLD. For example, while TRAF2 and TRAF6 undergo "activating" K63-linked autoubiquitination, K48-linked polyubiquitin chains play an important role in NF-B signaling by targeting phosphorylated I B proteins for degradation in the 26S proteasome (21)(22)(23). To assess whether YopJ can promote the cleavage of K48-linked polyubiquitin chains as well as K63-linked chains, which are attached to TRAF2 and TRAF6, we expressed TRAF6 in cells to activate NF-B signaling and used the proteasome inhibitor MG132 to trap the predominantly K48-linked polyubiquitinated proteins that would otherwise be degraded and not detected. We found that WT YopJ, but not CYLD, was able to block the accumulation of ubiquitinated proteins that occurred in cells treated with MG132 (Fig. 3 C). Next, we focused directly on K48-and K63-linked polyubiquitin chains by cotransfecting cells with TRAF6 and HA-tagged ubiquitin that had every lysine residue except K48 or K63 mutated to arginine (Fig. 3 D). MG132 treatment was used again to prevent the degradation of cellular proteins modified by K48-linked polyubiquitin chains. Western blotting with anti-HA antibodies revealed that YopJ interfered with the accumulation  of proteins with K48-or K63-linked polyubiquitin chains, whereas CYLD only blocked the accumulation of proteins with K63-linked polyubiquitin chains (Fig. 3 D). These data indicate that YopJ can promote the cleavage of both K48and K63-linked polyubiquitin chains and, as such, has much broader target specificity than CYLD. This difference might account for the ability of YopJ to inhibit NF-B signaling in more settings than CYLD. For example, YopJ can inhibit NIK-induced NF-B activation but CYLD cannot (Fig. 3  B). Such promiscuity in a bacterial enzyme would enable efficient shutdown of the multiple signaling pathways that contribute to the host immune response. Similar substrate promiscuity is associated with another Yersinia virulence factor, the tyrosine phosphatase YopH (24)(25).
YopJ is essential for Y. pseudotuberculosis -induced macrophage apoptosis and is important for establishing a systemic infection in mice as indicated by increased clearance of a yopJ mutant 4 d after an oral infection in mice (2,3,26). Thus, to extend our findings in 293T cells, we tested the importance of the deubiquitinating activity associated with YopJ in the relevant setting of an actual bacterial infection. Mouse macrophages were infected with either WT Y. pseudotuberculosis or a YopJ-null strain of Y. pseudotuberculosis that had been complemented with the catalytic cysteine YopJ mutant C172A. Western blot analysis of whole cell lysates was performed at different times after infection (Fig. 4 A). At 5 min after infection, phosphorylated I B ␣ was observed in macrophages infected with WT YopJ Y. pseudotuberculosis as well as in macrophages infected with YopJ C172A Y. pseudotuberculosis , suggesting that both bacteria initially induced similar activation of the IKK complex, presumably through engagement of surface Toll-like receptors (Fig. 4 A, top; compare lanes 1, 2, and 6). Interestingly, the levels of phosphorylated I B ␣ remained elevated even at 60 min after infection with WT YopJ Y. pseudotuberculosis, whereas levels of phosphorylated IB␣ in cells infected with YopJ C172A Y. pseudotuberculosis were greatly decreased by 30 min after infection (Fig.  4 A, top; compare lanes 4 and 8). Immunoblotting for total IB␣ levels confirmed that IB␣ was degraded in cells infected with YopJ C172A Y. pseudotuberculosis but not in cells infected with WT YopJ (Fig. 4 A, middle; compare lanes 4 and 8). Phosphorylation of IB␣ normally is a prerequisite for its ubiquitination and degradation by the proteasome (5), so we investigated whether phosphorylated IB␣ was not degraded in cells infected with WT YopJ Y. pseudotuberculosis because it was deubiquitinated efficiently by YopJ. To test this hypothesis, we treated macrophages with the proteasome inhibitor MG132 before infection to trap and detect the otherwise highly labile ubiquitinated IB␣. At different times after infection, IB␣ was immunoprecipitated from boiled lysates and immunoblotted with ubiquitin antibodies (Fig. 4 B). High molecular weight bands, consistent with polyubiquitinated IB␣, were observed by 5 min after infection in cells infected with WT YopJ or YopJ mutant C172A bacteria. Notably, these higher molecular weight species then disappeared rapidly from cells infected with WT YopJ Y. pseudotuberculosis despite the presence of MG132 (Fig. 4  B). By contrast, the polyubiquitinated IB␣ was still evident in cells infected with YopJ C172A Y. pseudotuberculosis at 60 min after infection. These results indicate that YopJ plays an essential role in reversing the ubiquitination of phosphorylated IB␣ during Yersinia infection. Consequently, NF-B transcription factors would stay sequestered in the cytoplasm by intact IB␣, thereby attenuating the antiapoptotic NF-B pathway (2,26,27). We propose that the reason the IKK complex can still be activated in macrophages infected with WT YopJ Y. pseudotuberculosis (Fig. 1 A), despite the fact that YopJ can promote the deubiquitination of such upstream components as TRAF2 and TRAF6 (Fig. 2 C), is that attachment of Yersinia to the macrophage surface activates NF-B signaling through Toll-like receptors (28) and perhaps additional cell surface receptors before the time that YopJ is injected into the macrophage by the bacteria's type III secretion system. Consistent with this notion, by immuno-gold labeling and electron microscopy, substantial levels of YopJ could not be detected in macrophages infected with Y. pseudotuberculosis until 15 min after infection (not depicted), a time when IB␣ is already phosphorylated (Fig. 4 A).
These results indicate that YopJ promotes deubiquitination, though it remains uncertain whether YopJ has intrinsic deubiquitinating activity or regulates ubiquitination indirectly via other proteins in cells. To demonstrate deubiquitinating activity for YopJ directly, we purified WT YopJ and YopJ mutant C172A from insect cells (Fig. 5 A) and incubated them with ubiquitin-7-amino-4-methylcoumarin (ubiquitin-AMC) in an in vitro deubiquitination assay (Fig. 5 B). Purified WT YopJ cleaved ubiquitin-AMC, but not SUMO-1-AMC, as judged by the release of fluorescent AMC, whereas YopJ mutant C172A was inactive against ubiquitin-AMC. Cleavage of SUMO-1-AMC under these experimental conditions was observed with recombinant SUMO protease 1 (Fig. S1, available at http://www.jem.org/cgi/content/full/ jem.20051194/DC1). These results indicate that YopJ possesses intrinsic deubiquitinating protease activity that requires cysteine 172. YopJ cleaved ubiquitin-AMC with a Km of 0.3ف uM, which is comparable with that of other known deubiquitinating proteases (29).
In sum, we provide the first direct evidence that a bacterial virulence factor, YopJ, is a deubiquitinating protease and have shown that this activity is responsible for YopJ-mediated inhibition of NF-B and MAPK signaling. Given that YopJ and its related proteins function as virulence factors in bacteria that cause: (a) plague or black death (Yersinia pestis), a disease of historical significance and of recent interest due to the threat of bioterrorism, (b) gastroenteritis (Y. pseudotuberculosis, Salmonella typhimurium), and (c) black rot in plants (Xanthomonas campestris), the finding that purified YopJ is a deubiquitinase provides a facile enzymatic assay for the eventual development of a therapeutic inhibitor. Additionally, it is likely that interfering with ubiquitin signaling in the host might be a common strategy used by pathogens to evade the host immune system during the course of infection.

MATERIALS AND METHODS
Transfection experiments. 293T cells were transfected with Lipofectamine 2000 (Invitrogen). Luciferase assays were performed as described previously (30). Ubc9 siRNA duplexes with 3Ј dTdT overhangs (Genentech, Inc.) or scramble II siRNA duplexes (Dharmacon) were transfected twice at 24-h intervals, with the cells being split to 80% confluency before the second transfection. Expression constructs were also included in the second transfection, and lysates were collected after an additional 36 h. Cells washed with PBS were lysed for 0.5 h at 4ЊC in 120 mM NaCl, 50 mM Hepes, pH 7.2, 1 mM EDTA, 0.1% NP-40, and complete protease inhibitor cocktail (Roche). Proteins were immunoprecipitated for 2-16 h at 4ЊC and then washed three times in lysis buffer. To assess ubiquitination, lysates were supplemented with 1% SDS (vol/vol) and heated at 90ЊC for 5 min. Samples were then diluted 10-fold with lysis buffer, and immunoprecipitations were performed.
Protease assays. WT YopJ, mutant YopJ, or SUMO protease 1 041ف( nM; LifeSensor Inc.) was incubated with ubiquitin-AMC or Sumo-AMC (0.25-7.5 M; Boston Biochem) in 50 mM Hepes, pH 8.0, and 1 mM DTT. Liberation of AMC at 23ЊC was monitored continuously in a microplate reader (excitation/emission wavelengths 380/460 nm; SpectraMax M2; Molecular Devices). Relative fluorescence units were converted to pmol AMC using a standard curve of relative fluorescence units versus AMC concentration. Kinetic constants (k cat , K m ) were calculated from Michaelis-Menten plots (V 0 versus [S]) with nonlinear regression analysis using Graph-Pad software and the assumption that 100% of purified YopJ was active.

Yersinia infections.
Yersinia strains were grown overnight with aeration in 2x YT medium at 26ЊC and then diluted 1:50 into 2x YT plus 20 mM sodium oxalate and 20 mM MgCl 2 to be grown for 2 h at 26ЊC and then 2 h at 37ЊC. 2 ϫ 10 6 RAW264.7 (ATCC TIB71) cells were seeded into sixwell plates at 37ЊC (5% CO2) for 15-18 h and infected at a multiplicity of infection of 50:1. Centrifugation at 165 g for 5 min synchronized the infection. 50 M MG132 was added 1 h before infection.
Online supplemental material. Fig. S1 shows the protease activity of recombinant SUMO protease 1 or YopJ against ubiquitin-AMC or SUMO1-AMC. It is available at http://www.jem.org/cgi/content/full/ jem.20051194/DC1. We thank Kim Newton for critical reading and editorial assistance, Karen O'Rourke for excellent technical assistance, and members of the Dixit lab for helpful discussion.
This work was supported by the National Institutes of Health grants P01AI063302 and U-19AI057229 to D.M. Monack.
The authors have no conflicting financial interests.


s2fieldsofstudy: Biology Medicine

text:
Phase II study of gemcitabine plus oxaliplatin as first-line chemotherapy for advanced non-small-cell lung cancer

This phase II study evaluated the response rate and tolerability of gemcitabine–oxaliplatin chemotherapy in non-small-cell lung cancer (NSCLC) patients. Chemonaive patients with stage IIIB or IV NSCLC received gemcitabine 1000 mg m−2 on days 1 and 8, followed by oxaliplatin 130 mg m−2 on day 1. Cycles were repeated every 21 days for up to six cycles. From February 2002 to May 2004, 60 patients were enrolled into the study in seven Italian institutions. We observed one complete response (1.7%) and 14 partial responses (23.3%), for an overall response rate of 25.0% (95% confidence interval, 14.7–37.9%). The median duration of response was 5.9 months (range 1.5–17.1 months). With a median follow-up of 6.7 months, median time to progressive disease and overall survival were 2.7 (range 1.9–3.4 months) and 7.3 months (range 7.2–8.6 months), respectively. The main grade 3–4 haematological toxicities were transient neutropenia in 11.7% and thrombocytopenia in 8.3% of the patients. Nausea/vomiting was the main grade 3–4 nonhaematological toxicity, occurring in 10.0% of the patients. Two (3.3%) patients developed grade 3 neurotoxicity. Our results show that gemcitabine–oxaliplatin chemotherapy is active and well tolerated in patients with advanced NSCLC, deserving further study, especially for patients not eligible to receive cisplatin.

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer death in the world (Greenlee et al, 2001). Platinum-based chemotherapy is considered the standard treatment for advanced disease, with cisplatin considered the most effective for NSCLC (Non-Small Cell Lung Cancer Collaborative Group, 1995). New agents, such as taxanes and vinorelbine, administered with a platinum derivative, have resulted in improved median and 1-year survival times when compared with either cisplatin alone or older platinum-based combinations in randomised trials (Le Chevalier et al, 1994;Wozniak et al, 1998;Bonomi et al, 2000;Sandler et al, 2000).
Gemcitabine's mild toxicity profile, both in terms of haematological toxicity and visceral side effects, and novel mechanism of action have encouraged its use in combination with traditional agents, such as cisplatin. Gemcitabine -cisplatin chemotherapy is considered one of the most active regimens for advanced NSCLC, with an overall response rate (ORR) of 22 -38% and median survival of 8.1 -9.8 months in phase III trials (Crinò et al, 1999;Scagliotti et al, 2002;Schiller et al, 2002). Although several phase III trials of new platinum-based doublets (Kelly et al, 2001;Schiller et al, 2002;Smit et al, 2003) have produced similar results, a recent meta-analysis showed an absolute 1-year survival benefit of 3.9% for gemcitabine -cisplatin chemotherapy when compared to other platinum-containing regimens (Le Chevalier et al, 2005).
Cisplatin has been the most widely used platinum derivative, but it is known to yield significant toxicities, including severe nausea and vomiting, and renal toxicity, requiring adequate hydration, ototoxicity and neuropathy. Oxaliplatin is a diaminocyclohexane platinum compound, with a mechanism of action similar to that of cisplatin (Woynarowski et al, 2000), that has shown activity against a variety of cancer types, including colon (de Gramont et al, 1997) and NSCLC (Monnet et al, 1998). Oxaliplatin has a more manageable toxicity profile than cisplatin, with no renal toxicity and a lower incidence of haematological and gastrointestinal toxicities. It lacks the nephrotoxicity of cisplatin and causes less myelotoxicity than carboplatin, the leading cisplatin analog, and it may be active in cisplatin-and carboplatin-resistant tumours (Chollet et al, 1996;Raymond et al, 1998b). The main toxicity associated with oxaliplatin is a generally reversible peripheral neuropathy (Raymond et al, 1998a).
Oxaliplatin has demonstrated synergy with gemcitabine, with a sequence dependency that favours the administration of gemcitabine, followed by oxaliplatin (Faivre et al, 1999). A phase I trial, conducted by Shibata et al (2001), investigated the maximum tolerated dose of gemcitabine -oxaliplatin in patients with solid tumours. This study showed that oxaliplatin 130 mg m À2 on day 1 could be safely administered with gemcitabine 1000 mg m À2 on days 1 and 8 every 21 days. Using different schedules, other investigators (Faivre et al, 2002;Mavroudis et al, 2003) confirmed that gemcitabine -oxaliplatin is feasible, with no dose-limiting toxicities, easy to administer on an outpatient basis, and shows promising activity in patients with NSCLC and ovarian cancer.
On the basis of these data, we designed a phase II, multicentre trial to evaluate the response rate and tolerability of gemcitabineoxaliplatin in untreated patients with stage IIIB or IV NSCLC, applying the same 3-week dosing schedule as per Shibata et al. Secondary end points were time to progressive disease (TtPD) and overall survival (OS).

Patient selection
Chemonaive patients with histologically or cytologically confirmed locally advanced or metastatic stage IIIB or IV NSCLC not amenable to surgery or radiotherapy with curative intent were eligible for this study. Other eligibility criteria included the presence of at least one measurable lesion; age X18 to p75 years; Karnofsky performance status (KPS) X70, life expectancy X12 weeks; adequate bone marrow reserve (absolute neutrophil count X2.0Â10 9 l À1 , platelet count X100Â10 9 l À1 ). Patients with asymptomatic brain metastases were eligible. No prior chemotherapy or immunotherapy or concomitant radiotherapy was allowed. Prior radiotherapy was acceptable if completed 4 weeks before study entry.
Exclusion criteria included inadequate liver function (bilirubin 41.5 times above normal range, aspartate transaminase (AST) and alanine transaminase (ALT) 43 times the normal range, or five times greater than the normal range in the presence of liver metastases); severe alteration of renal functionality (serum creatinine 42 Â the upper limit of normal (ULN)); no second malignancies (except for carcinoma of the cervix uteri in situ and squamous cell carcinoma of the skin); peripheral neuropathy; untreated superior vena cava syndrome; hypercalcaemia requiring intravenous (i.v.) treatment; the presence of uncontrolled cardiac disease, uncontrolled diabetes mellitus, infections or other medical conditions that could have interfered with the trial.
Written informed consent was obtained from each patient before entering the study. The study was approved by the local Ethics Committee and was conducted in accordance with ethical principles stated in the most recent version of the Declaration of Helsinki or the applicable guidelines on good clinical practice, whichever represented the greater protection of the individual.

Treatment plan and dose adjustments
Gemcitabine (Gemzar s , Eli Lilly and Company, Indianapolis, IN, USA) 1000 mg m À2 was administered as a 30-min i.v. infusion on days 1 and 8 of a 21-day cycle. Oxaliplatin (Eloxatin s , Sanofi-Synthelabo, Paris, France) 130 mg m À2 was administered as a 2-h i.v. infusion on day 1, following gemcitabine. All patients were scheduled to receive at least two cycles of therapy, and up to six cycles if there was no evidence of disease progression. Treatment was stopped early in cases of patient refusal, severe toxicity, documented progressive disease (PD) or pregnancy. Patients with PD after two or four cycles or with stable disease after four cycles of chemotherapy were withdrawn from the study. Full supportive therapy, corticosteroids, anticonvulsants and antibotics were given as needed. Antiemetic premedication included 5-HT 3 antagonists. No routine use of haematopoietic growth factors was planned. No prophylactic antibiotics were used. All patients were treated on an outpatient basis.
Dose adjustments during treatment were based on haematological and nonhaematological toxicities. On day 1, if neutrophil count was o1.5 Â 10 9 l À1 and/or platelet count was o100 Â 10 9 l À1 , chemotherapy doses were delayed (for up to 2 weeks) and doses were reduced by 25% to allow recovery from haematological toxicity. On day 8, for a neutrophil count o1.0 and/or platelets o75, the gemcitabine dose was omitted, and the cycle continued with one gemcitabine dose not given. Patients not recovering from haematological toxicity (neutrophil count 41.0 and platelets 475) within 2 weeks were withdrawn from the trial. Doses were reduced by 25% for any grade 3 nonhaematological toxicity (excluding nausea, vomiting and alopecia). Treatment was discontinued in the event of grade 4 or frequent grade 3 nonhaematological toxicity. For grade 2 -4 neurological toxicity, oxaliplatin treatment was delayed until the patient recovered to grade 1, then the dose was reduced by 25%. If no recovery to grade 1 was achieved in 3 weeks, the patient was discontinued from the trial.
For hepatic toxicity, the day-1 gemcitabine dose was reduced by 25% for AST/ALT 1.6 to 5.0 Â ULN, alkaline phosphatase p5.0 Â ULN and bilirubin oULN. Treatment was delayed for up to 2 weeks for AST/ALT 45 Â ULN or alkaline phosphatase 45 Â ULN or bilirubin 4ULN. The day-1 gemcitabine dose was omitted for a total bilirubin 41.5 ULN. For nephrotoxicity, gemcitabine doses were reduced by 25% for serum creatinine 41.5 -2.0 ULN and creatinine clearance X50 ml min À1 . Treatment was delayed for serum creatinine 41.5 UNL and creatinine clearance o50 ml min À1 or serum creatinine greater than 42.0 UNL with any creatinine clearance value.

Baseline and treatment assessments
Pretreatment evaluation included physical examination; KPS; chest X-ray; brain, thoracic and abdominal computer tomography scan (CT scan); bronchoscopy (if not performed at the time of diagnosis); bone scan; electrocardiogram; complete blood count and blood chemistry with liver function tests and creatinine clearance. On days 1 and 8, a physical exam (including weight) was performed, and KPS and blood count were assessed. All measurable and evaluable lesions were assessed by the same method used at baseline. Response to therapy was assessed every two cycles with clinical and/or radiological tumour assessment.
In responding patients, a confirmatory assessment was repeated after at least 4 weeks, according to the RECIST criteria (Therasse et al, 2000). Assessment of TtPD was determined by measuring the time interval from the beginning of treatment until the first documentation of progression or death due to any cause. Survival (OS) was determined by measuring the time interval from the beginning of the treatment to the date of death or last contact. Toxicity was evaluated according to NCI criteria (Common Toxicity Criteria, 1993). No quality of life questionnaire was used in the present study.

Statistical plan
A two-stage design was used for the study. Using the Simon (1989) hypothesis, assuming a response rate of 40%, a probability of error of 5% and a power of 90%, a total of 54 patients were to be enrolled, with at least five responses to be noted in the first 19 patients. Treatment was considered effective if at least 16 objective responses were observed. Every patient included in the study was considered evaluable (intent-to-treat analysis). Response rates, including 95% confidence intervals (CIs), were calculated on an intent-to-treat basis. Time-to-event end points were calculated using the Kaplan -Meier method, with the appropriate censoring (Kaplan and Meier, 1958).

Patient characteristics
From February 2002 to May 2004, a total of 60 patients were enrolled in this trial in seven Italian centres. Patients' characteristics at baseline are listed in Table 1. The majority of patients were male (71.7%), with a median age of 61 years (range 36 -74). Performance status was 100 in 46.7% of patients; 51 (85.0%) patients had stage IV and nine (15.0%) had stage IIIB disease. Histology was predominantly adenocarcinoma (55.9% of the patients).

Efficacy
As per the ITT analysis, the ORR was 25.0% (95% CI, 14.7 -37.9%), including one complete response (CR: 1.7%) and 14 partial responses (PRs: 23.3%, Table 2). Two patients were not assessable for response because they discontinued the trial after the first cycle, one for heart attack and the other for cardiac failure; neither event was related to the study drugs. The median duration of response was 5.9 months (range 1.5 -17.1 months). With a median follow-up time of 6.7 months, the median TtPD was 2.7 (range 1.9 -3.4 months), median OS time was 7.3 months (range 7.2 -8.6 months) and 1-year survival was 36% (Figures 1, 2).

Treatment administration
A total of 209 chemotherapy courses were administered, with a median of 3.9 cycles per patient (range 1 -6), and 16 (26.7%) patients received all six cycles. The planned dose intensity was 666.6 mg m À2 week À1 for gemcitabine and 43.3 mg m À2 week À1 for oxaliplatin. Dose intensity for all 60 patients was 615 mg m À2 week À1 for gemcitabine and 42.4 mg m À2 week À1 for oxaliplatin, with 92.3% of the planned gemcitabine dose and 97.9% of the planned oxaliplatin dose delivered.

Toxicity
The main haematological toxicities (Table 3) were transient grade 3 -4 neutropenia observed in seven (11.7%). patients, grade 3 -4 thrombocytopenia not requiring platelet transfusion in five (8.3%) patients and grade 3 anaemia in one (1.7%) patient. No neutropenic fever or bleeding episodes were recorded. Nonhaematological toxicity was generally manageable (Table 4). Grade 3 nausea/vomiting occurred in five (8.3%) and grade 4 occurred in one (1.7%) patients. Two (3.3%) patients developed grade 3 neurotoxicity requiring oxaliplatin dose reduction. Two (3.3%) patients had grade 4 cardiovascular toxicity consisting of extrasystoles and atrial flutter. A total of 25.0% of the patients displayed grade 1 -2 pulmonary symptoms, such as dyspnea, but this was due to disease progression. Grade 1 nephrotoxicity occurred in one (1.7%) patient. Two serious adverse events were reported during the study. One patient died after the first course due to heart attack and the other patient died due to cardiac failure. Neither of these deaths was considered treatment-related.

DISCUSSION
Our study confirmed the promising activity and favourable toxicity profile of gemcitabine -oxaliplatin in patients with stage IIIB/IV NSCLC. We observed an ORR of 25.0%, with one CR and 14 PRs. The median duration of response was 5.9 months (range 1.5 -17.1 months). Median TtPD was 2.7 months (range 1.9 -3.4 months) and median OS was 7.3 months (range 7.2 -8.6 months).
The 25% ORR observed in the present study seems to be not different from the 20 -22% response rate obtained with gemcitabine when used as single agent (Abratt et al, 1994;Gatzemeier et al, 1996). However, although our study population was relatively young and with good performance status, a large percentage of stage IV patients was enrolled, including individuals with unfavourable prognostic factors, such as brain metastases.
Two trials have evaluated the activity and toxicity of gemcitabine -oxaliplatin in NSCLC patients (Faivre et al, 2002;Franciosi et al, 2003). Both trials included pretreated patients, and response rates ranged from 16 to 34%. The 16% response rate reported in the Franciosi trial was lower than that reported in our study (25.0%), probably because our study was conducted only in chemonaive patients. In the study conducted by Faivre et al.,12 of the 35 patients (34.2%) enrolled in the trial responded to the therapy, including two platinum-resistant patients. These results are better than those observed in our trial, probably because of the different characteristics of our cohort, which included a larger percentage of stage IV disease (85% in our study vs 71%), and a lower percentage of patients with a performance status of 100 (46.7% in our study vs 85.7%).
The response rate observed with gemcitabine -oxaliplatin in the present trial was also comparable to the activity of oxaliplatinbased combinations observed in other phase II studies. Oxaliplatin has been evaluated in combination with vinorelbine (Monnet et al, 2001) and in combination with taxanes (Winegarden et al, 2004), producing response rates of 19 and 37%, respectively.
One of the most important findings of our trial is the low toxicity profile demonstrated by gemcitabine -oxaliplatin. In phase II trials, single-agent gemcitabine was administered at the dose of 1000 -1250 mg m À2 on days 1, 8 and 15 (Anderson et al, 1994;Gatzemeier et al, 1996;Le Chevalier et al, 1997), or at the dose of 1250 mg m À2 on days 1 and 8 when used in combination with cisplatin (Castellano et al, 1998). Planned dose intensity of gemcitabine in our study was lower than those reported by others (Anderson et al, 1994;Gatzemeier et al, 1996;Le Chevalier et al, 1997;Castellano et al, 1998), providing an additional explication for the excellent toxicity profile demonstrated by this regimen. We used the 130 mg m À2 dose per cycle for oxaliplatin and 2000 mg m À2 dose per cycle for gemcitabine evaluated in other reports (Franciosi et al, 2003;Mavroudis et al, 2003), and further demonstrated that the haematological and nonhaematological toxicities with this regimen are generally mild or moderate. The tolerability of the regimen is underscored by the fact that we were able to deliver full doses of the planned therapy to the majority of patients. The main grade 3 -4 haematological toxicities were transient neutropenia and thrombocytopenia (11.7 and 8.3% of patients, respectively). Nausea/vomiting was the main grade 3 -4 nonhaematological toxicity, occurring in six (10.0%) patients. Only two (3.3%) patients developed significant (grade 3) neurotoxicity.
The low incidence of side effects in our trial is of particular relevance to clinical practice because it suggests that gemcitabineoxaliplatin could be used in patients ineligible for standard platinum-based therapy, such as the elderly or unfit. At the present time, single-agent therapy is the standard treatment for elderly or unfit patients (Gridelli et al, 2003), but there is clear evidence that two-drug combinations are better than single-agent therapy (Sederholm, 2002;Lilenbaum et al, 2005), and platinum-based chemotherapy should be considered for selected elderly patients (Bunn and Lilenbaum, 2003).
In conclusion, our findings further demonstrate that gemcitabine -oxaliplatin is active and well tolerated in patients with advanced NSCLC. The favourable toxicity profile of this regimen also justifies its use in patients in whom cisplatin may not be feasible, such as the elderly or unfit. Oxaliplatin hopefully will  Neutropenia 6 (10.0) 1 (1.7) 16 (7.7) 1 (0.5) Thrombocytopenia 4 (6.7) 1 (1.7) 10 (4.8) 1 (0.5) Anaemia 1 (1.7) 0 1 (0.5) 0 Leukopenia 1 (1.7) 0 1 (0.5) 0   become an alternative to cisplatin whose toxicity profile is not acceptable for many patients with NSCLC. In the era of targeted therapies, this combination deserves to be studied further in association with new agents, such as the antivascular endothelial growth factor bevacizumab, or the monoclonal antibody antiepidermal growth factor receptor cetuximab.


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Myeloid and plasmacytoid dendritic cells transfer HIV-1 preferentially to antigen-specific CD4+ T cells

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of T cell immunity against pathogens such as human immunodeficiency virus (HIV)-1. At the same time, HIV-1 replication is strongly enhanced in DC–T cell clusters, potentially undermining this process. We found that immature CD123+ plasmacytoid DCs (PDCs) and CD11c+ myeloid DCs (MDCs) were susceptible to both a CCR5- and a CXCR4-using HIV-1 isolate in vitro and were able to efficiently transfer that infection to autologous CD4+ T cells. Soon after HIV-1 exposure, both PDCs and MDCs were able to transfer the virus to T cells in the absence of a productive infection. However, once a productive infection was established in the DCs, newly synthesized virus was predominantly spread to T cells. HIV-1 exposure of the MDCs and PDCs did not inhibit their ability to present cytomegalovirus (CMV) antigens and activate CMV-specific memory T cells. As a result, both PDCs and MDCs preferentially transmitted HIV-1 to the responding CMV antigen–specific CD4+ T cells rather than to nonresponding T cells. This suggests that the induction of antigen-specific T cell responses by DCs, a process crucial to immune defense, can lead to preferential HIV-1 infection and the deletion of responding CD4+ T cells.

HIV-1 infection is associated with a gradual loss of CD4 ϩ T cells and severe dysfunction of the immune system. Of particular note, HIV-1 preferentially infects CD4 ϩ T cells specific for HIV-1 antigens, as compared with T cells with other antigen specificities within infected individuals (1). Resting CD4 ϩ T cells are the major reservoir for HIV-1 infection in vivo (2)(3)(4). DCs are important for stimulating antigenspecific CD4 ϩ T cell responses from the resting memory T cell pool, as well as priming naive T cells (5). Because DCs can be productively infected with HIV-1, and infected DCs can be found in mucosal tissues soon after viral transmission (6,7), they could potentially serve as Trojan horses, delivering the signals necessary for inducing CD4 ϩ T cell responses while simultaneously delivering a lethal dose of HIV-1 to those responding T cells. In fact, the DC-T cell microenvironment has been described as an "explosive" site for HIV-1 propagation, as activated T cells become highly susceptible to HIV-1 and thereby further enhance viral replication (8)(9)(10)(11)(12). Still, it is unknown whether DCs are as central to the pathogenesis of HIV-1 infection as they are for generating effective, adaptive anti-HIV-1 immunity.
Although studies of the role of DCs in HIV-1 infection have substantially intensified over the last decade, most knowledge has been revealed using in vitro monocyte-derived DCs to model this process, often using allogeneic T cell lines as a surrogate for antigen-specific T cells. To provide a more physiologic system, we modeled the spread of HIV-1 during specific CMV antigen presentation by DC subsets to autologous CMV-specific CD4 ϩ T cells isolated directly from blood. We investigated CD123 ϩ plasmacytoid DCs (PDCs) and CD11c ϩ myeloid DCs (MDCs), both of which have previously been found to be susceptible to HIV-1 infection (13)(14)(15)(16).
Here, these subsets of DCs were infected with HIV-1 ex vivo and were subsequently co-cultured with autologous CD4 ϩ T cells. Using flow cytometric analyses, we documented that MDCs and PDCs displayed differential susceptibility to the R5-and X4-HIV-1 strains Both DC subsets differentiated partially in response to HIV-1 exposure alone, as found by up-regulation of CD40, CD80, CD83, and CD86 ( Fig. 1 E). However, Toll-like receptor (TLR)7/8 ligation with R-848 led to complete maturation exceeding the differentiation induced by HIV-1 exposure alone ( Fig. 1 E). We did not find that R-848-induced activation of HIV-1-exposed MDCs or PDCs led to a different cell surface phenotype than the activation of uninfected DCs (16).

HIV-1-infected DCs transfer the virus to autologous CD4 ؉ T cells
Prior studies have shown that conjugates of DCs and T cells are predominant sites for HIV-1 replication both in vivo and in vitro (8)(9)(10)(11)(12). We examined the specific transfer of HIV-1

Figure 1. Productive HIV-1 BaL and HIV-1 IIIB infection in MDCs and
PDCs. (A) Sorted CD11c ϩ MDCs (red) and CD123 ϩ PDCs (blue) were analyzed for their expression of CD4, CCR5, and CXCR4 after overnight culture. MDCs (B) and PDCs (C) were exposed to HIV-1 BaL or HIV-1 IIIB in the presence or absence of AZT for 72 h using medium as a control. The level of HIV-1 infection was determined by intracellular p24 staining and flow cytometry. Any contaminating monocytes were excluded from the analysis by gating on the cells that expressed CD11c (MDCs) or CD123 (PDCs), but not CD14. The numbers in each gate represent the percentages of p24 ϩ cells out of all cells. DCs from 2 of 14 representative donors are shown. (D) MDCs (black bars) and PDCs (white bars) from 12 individual donors (mean Ϯ SEM) were exposed to HIV-1 BaL or HIV-1 IIIB for 72 h and the supernatants were analyzed for IFN-␣ content by ELISA. Significant differences between the groups were assessed by Wilcoxon's paired t test (***, P Յ 0.0005). (E) Histograms of the cell surface expression of the costimulatory molecule CD86 on MDCs and PDCs are shown for one representative donor at 72 h (gray region, medium; black line, HIV-1 BaL ; green line, R-848 stimulation).

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between HIV-1-exposed subsets of DCs and autologous CD4 ϩ T cells during antigen presentation. To establish such an experimental system, we first evaluated the time required for the transmission of HIV-1 from infected DCs to uninfected autologous T cells. MDCs or PDCs, isolated from healthy CMV-seropositive donors, were exposed to HIV-1 for 72 h, washed thoroughly, and incubated briefly with inactivated CMV antigen lysate or Staphylococcal enterotoxin B (SEB), and then co-cultured with sorted CD4 ϩ T cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). The proliferation of CMV antigen-specific memory T cells responding to DCs presenting processed CMV peptides, or of T cells activated thorough TCR V ␤ -restricted SEB cross-linking between DCs and T cells, was assessed at different time points after co-culture (1.5, 3.5, and 7.5 d) by flow cytometry. To analyze only T cells, gates were set on CFSE-labeled T cells excluding CD11c ϩ MDCs or CD123 ϩ PDCs. HIV-1 p24 expression and CFSE dilution in the T cells were thereafter assessed and compared with T cells cocultured with uninfected DCs (Fig. 2). At 1.5 d after co-culture, there was no T cell division and no or very low intracellular HIV-1 p24 in the T cells (Fig. 2 A). At 3.5 d, T cells expressing HIV-1 p24 were detected and were more frequent in cultures where T cell activation and division had been stimulated (CMV or SEB) than in those where no stimulation had occurred (Fig. 2 B). The frequency of p24 ϩ T cells increased over time. At 7.5 d after co-culture, a large percentage (ranging from 3 to 19%) of the T cells in the cultures that had received CMV or SEB stimulation expressed p24, whereas T cell infection in the nonstimulated cultures remained minimal (Fig. 2 C). HIV-1-infected MDCs and PDCs were able to activate CMV-specific T cells or SEBstimulated T cells to proliferate to a similar extent as uninfected donor-matched DCs (Fig. 2, C and D). Both HIV-1 BaL -and HIV-1 IIIB -exposed MDCs or PDCs were able to transfer the virus to CD4 ϩ T cells as p24 ϩ T cells were detected after 3.5 d of co-culture, irrespective of which virus isolate or DC subset was used. A time period of 3.5 d for cell co-culture was therefore chosen for further studies. To ensure that virus saturation had not been reached in our culture system, we exposed DCs to 10-fold dilutions of the virus stocks before co-culture with T cells. Proportionally fewer T cells became infected when co-cultured with DCs that had been exposed to lower amounts of either HIV-1 BaL (Fig. 2 E) or HIV-1 IIIB (unpublished data). Differential transfer of HIV-1 BaL and HIV-1 IIIB from MDCs and PDCs to CD4 ؉ T cells As mentioned in Productive HIV-1...PDCs section, we found that MDCs were more susceptible to R5-using HIV-1 BaL than to X4-using HIV-1 IIIB , whereas no preference was found for PDCs ( Fig. 1, B and C). We therefore asked how efficiently the different virus strains were transferred from the DC subsets to T cells during co-culture. MDCs were shown to efficiently transfer both HIV-1 BaL and HIV-1 IIIB to T cells (Fig. 3, A and E). We found no significant difference in the efficiency of transfer and/or propagation of the different virus isolates by the MDCs (P ϭ 0.42; Fig. 3 E). In contrast, HIV-1 IIIB was more efficiently transferred to and propagated in T cells by PDCs, as compared with HIV-1 BaL (P ϭ 0.03; Fig. 3, B and E). Production of p24 in infected T cells represents a late stage of the virus replication cycle and may not reflect the absolute amount of virus that is transferred from DCs to T cells (20). In addition, the infection of T cells that appeared 3.5 d after co-culture may represent a direct Sorted CD11c ϩ MDCs were exposed to HIV-1 BaL for 72 h, washed thoroughly, and incubated briefly with CMV or SEB before co-culture with sorted and CFSE-labeled autologous CD4 ϩ T cells. (A-C) Co-cultures were analyzed for T cell proliferation (CFSE dilution) and frequency of HIV-1 infection (intracellular p24 staining) after 1.5, 3.5, and 7.5 d. The numbers in each gate indicate the percentages of p24 ϩ cells out of all cells. (D) As a control, uninfected MDCs were co-cultured with T cells in the presence or absence of CMV or SEB to measure background staining for p24 and examine the rate of proliferation in the absence of HIV-1. The graphs show CD4 ϩ T cells gated on the presence of CFSE labeling, excluding single CD11c ϩ cells to leave out MDCs from the analysis. One representative experiment of three is shown. The numbers in each gate indicate the percentages of p24 ϩ cells out of all cells. (E) PDCs (open squares) and MDCs (filled squares) were exposed to different doses of HIV-1 BaL for 72 h before co-culture with T cells in the presence of SEB. The frequency of p24 ϩ T cells was again measured after 3.5 d of co-culture. One representative experiment of four is shown. transfer of the virus from infected DCs, but may also represent T cell to T cell propagation of HIV-1 after an initial transfer of the virus from DCs to T cells. To distinguish between these possibilities, we added the protease inhibitor Indinavir to the co-cultures (see Materials and methods). Indinavir allows the formation of p24 in infected cells, but blocks the production of infectious viral particles and the subsequent spread of the virus between CD4 ϩ T cells. The addition of Indinavir led to a reduction of p24 ϩ T cells after coculture with HIV-1-infected DCs, indicating that HIV-1 is primarily propagated by T cell to T cell spread after the initial transfer from DCs in this in vitro system (Fig. 3, A-D). With Indinavir present in the co-culture, significantly higher frequencies of p24 ϩ T cells were found after co-culture with HIV-1 BaL -as compared with HIV-1 IIIB -exposed MDCs (P ϭ 0.02; Fig. 3, C and E). In contrast, HIV-1 IIIB -exposed PDCs were again able to infect a higher number of T cells as compared with HIV-1 BaL -exposed PDCs (P ϭ 0.03; Fig. 3, D and E). HIV-1 IIIB was transferred equally well by PDCs and MDCs (P ϭ 0.81). In contrast, HIV-1 BaL was more efficiently transferred to T cells by MDCs than PDCs (P ϭ 0.008; Fig. 3, C-E). Although neither of the virus isolates appeared to reach virus saturation at the inoculum concentration (1 g p24/ml) used for this comparison, it is still possible that different virus strains may have different transfer efficiencies from DC subsets to T cells at different virus titers. Several factors may therefore influence the selection and propagation of virus strains taking place in vivo.

Different pathways for transfer of the virus can be used by MDCs and PDCs
There are at least two pathways for the binding of HIV-1 by DCs. In addition to the conventional CD4-and coreceptormediated infection, HIV-1 can also bind to C-type lectin receptors such as DC-SIGN (CD209), which may lead to internalization by DCs (21)(22)(23)(24)(25)(26)(27). To examine whether a productive infection by isolated PDCs and MDCs is required to transfer the virus to T cells, we supplemented the culture media with AZT during the 72 h of HIV-1 exposure of DCs. AZT was thereafter washed out together with a residual virus before the DCs were co-cultured with autologous CD4 ϩ T cells for 3.5 d. Co-culture of T cells with AZT-treated DCs resulted in no p24 expression in the T cells (Fig. 4, A-D). This was true for both PDCs and MDCs exposed to either HIV-1 BaL or HIV-1 IIIB (Fig. 4, A-D). These data show that longer term HIV-1 transfer by PDCs and MDCs to T cells is dependent on the productive infection of DCs. It has been shown that the time of virus exposure influences the pathway for transfer of the virus from DCs to T cells (27). To examine whether MDCs and PDCs have the ability to transfer the early-captured virus in the absence of infection, we limited the virus exposure time to 6 h, washed, and then added T cells for 3.5 d. Both MDCs and PDCs were able to pass the virus to T cells using this shorter virus exposure period (Fig. 4, E and F). Thus, in line with previous reports using other types of DCs, the MDCs and PDCs isolated from blood also transfer HIV-1 to T cells in the absence of a productive infection of the DCs soon after virus exposure.
DCs are predominantly of an immature phenotype when they first encounter HIV-1 at peripheral sites, but are mainly mature when they are located in lymph nodes to induce T cell activation and, presumably, the transfer of HIV-1. Therefore, to mimic the in vivo situation, immature MDCs and PDCs were exposed to HIV-1 for 72 h and stimulated with R-848 for the final 24 h to induce differentiation of DCs. R-848 stimulation led to a phenotypic maturation of both CD123 ϩ PDCs (B and D) were exposed to HIV-1 BaL or HIV-1 IIIB for 72 h, washed, and co-cultured with autologous CFSE-labeled CD4 ϩ T cells with SEB for 3.5 d in the absence (A and B) or presence (C and D) of the protease inhibitor Indinavir. Cultures were analyzed for T cell proliferation (CFSE dilution) and frequency of HIV-1 infection (intracellular p24 staining). The graphs show CD4 ϩ T cells gated on size and lack of CD11c or CD123 expression to exclude DCs from the analysis. The numbers in each gate indicate the percentages of p24 ϩ cells out of all cells. One representative experiment of at least five is shown. (E) The graphs show the frequency of p24 ϩ T cells (mean Ϯ SEM) after co-culture with MDCs (black bars) or PDCs (white bars) exposed to the respective virus strains. The co-cultures were performed in the absence or presence of Indinavir, as indicated. Significant differences between the groups were assessed by Mann-Whitney's unpaired t test (*, P Յ 0.05).

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MDCs and PDCs ( Fig. 1 E; reference 17). Maturation did not lead to increased viral production in either MDCs or PDCs, as measured by intracellular p24 expression (16). Despite this, mature MDCs more efficiently transferred and/or induced propagation of both HIV-1 BaL and HIV-1 IIIB in T cells than in donor-matched immature MDCs (P ϭ 0.007 and P ϭ 0.03, respectively; Fig. 4, A and B). There was no increase in the viral transfer of either of the isolates after maturation of PDCs (P ϭ 0.62 and P ϭ 0.84, respectively; Fig. 4, C and D). As for immature DCs, viral transfer to T cells by mature DCs did not occur when each of the DC subsets had been exposed to AZT during the 72-h infection period (Fig. 4

, A-D).
MDCs and PDCs preferentially transfer HIV-1 to antigenspecific CD4 ؉ T cells Viral transfer from DCs to T cells has been shown to occur within an "infectious synapse" formed between a DC and an adjacent CD4 ϩ T cell (27)(28)(29)(30). Still, it is not known if T cells become infected when DCs form a tight cluster with T cells and the immunological synapses between the cells also serve as infectious synapses. We have previously shown that HIV-1-specific CD4 ϩ T cells are preferentially infected by HIV-1 in vivo (1). This would suggest that antigen-specific T cells responding to a given antigen presented by an HIV-1infected DC would become infected more frequently than T cells not responding to specific signals transmitted by the DC. We investigated the relationship between a DC-mediated HIV-1 infection and the specific activation of CD4 ϩ T cells in more detail. Again, the HIV-1-infected DCs were incubated with CMV antigens and then co-cultured with autologous CD4 ϩ T cells. To ensure that HIV-1-infected T cells were infected exclusively by the DCs and not indirectly by T cell to T cell spread in the co-culture, the protease inhibitor Indinavir was added to the co-cultures. We initially examined the frequency of p24 ϩ T cells or CD4 ϩ T cells that did or did not produce the effector cytokines IFN-␥, TNF-␣, and/or IL-2 in response to CMV antigen presented by the DCs (Fig. 5 A). In addition, as displayed by CFSE dilution, we gated on each cell division within the cytokineproducing and noncytokine-producing T cells. We found significantly higher frequencies of p24 ϩ T cells among the cytokine-producing T cells as compared with the noncytokine-producing T cells (P Ͻ 0.0001). This indicates that among the T cells present in the co-cultures, the CMV-specific T cells that were responding to CMV antigens presented by DCs were most likely to become HIV-1 infected. The highest proportions of p24 ϩ cells were found in undivided T cells or T cells that had divided only once. Because induction of cytokine production occurs rapidly after memory T cell activation and before proliferation (31), this indicates that a majority of the p24 ϩ T cells were recently both activated and infected after their encounter with CMV antigen-presenting and/or HIV-1-infected DCs. Productive infection of T cells may lead to death before division and, therefore, it is not surprising that the frequency of infected T cells decreased in later divisions. In addition, HIV-1 Vpr causes cell cycle arrest and may also explain why most of the p24 ϩ T cells had not divided. Next, we examined whether p24 ϩ or p24 Ϫ T cells most often colocalized with the production of effector cytokines (Fig. 5 B). Again, each cell division was analyzed. There was more than twice the frequency of cytokine-producing CD4 ϩ T cells among the p24 ϩ cells than p24 Ϫ T cells. The greatest induction of preferential infection of antigen-specific cells occurred in the nondivided T cell population (Fig. 5, A and B). We also examined all the responding T cells in the co-cultures, i.e., both divided and cytokine-producing T cells (Fig. 5 C). Responding CMV-specific T cells were more frequently infected than nonresponding T cells (P Ͻ 0.0001; Fig. 5 C). The HIV-1-infected p24 ϩ cells within the responding T cells were more likely to express cytokines as compared with the p24 Ϫ T cells. The same pattern was observed when each and CD123 ϩ PDCs (C and D) in the presence or absence of AZT were exposed to HIV-1 BaL (A and C) or HIV-1 IIIB (B and D) for 72 h with or without R-848 stimulation during the final 24 h. DCs were washed, pulsed with CMV or SEB, and co-cultured with autologous CD4 ϩ CFSE-labeled T cells for 3.5 d. The frequency of p24 ϩ T cells after co-culture was analyzed by flow cytometry. The graphs show the percentage of p24 ϩ CD4 ϩ T cells gated on the presence of CFSE labeling and on size, excluding single CD11c ϩ /CD123 ϩ DCs in 6-11 individual donors. The mean frequency of p24 expression is indicated in each column. Significant differences between the groups were assessed by Mann-Whitney's unpaired t test (**, P Յ 0.005; *, P Յ 0.05). (E) MDCs (black bars) and PDCs (white bars) were exposed to HIV-1 BaL for 6 or 72 h in the absence or presence of AZT. DCs were then washed and co-cultured with T cells in the presence of SEB for 3.5 d. The graphs show the frequency of p24 ϩ T cells (mean Ϯ SEM) after co-culture in three donors. DC subset exposed to either HIV-1 BaL or HIV-1 IIIB was cocultured with T cells. This general trend-that HIV-1 infection was most frequent in responding T cells-was true both in the presence of CMV or SEB using either immature or mature DCs. This indicates that the majority of the infected T cells had interacted with an HIV-1-infected DC that simultaneously presented antigen to which the TCR of the T cells had specificity. Alternatively, the responding antigenspecific T cells received an HIV-1 infection from HIV-1infected DCs that were in close proximity to the antigenpresenting DCs.
To confirm our findings, we isolated fractions of responding and nonresponding T cells after co-culture with infected DCs using flow cytometry. The different T cell fractions (undivided cytokine producing; dividing [including all divisions] cytokine producing; dividing noncytokine producing; and undivided noncytokine producing) were analyzed for their content of HIV-1 gag DNA by real time PCR (Fig. 6 A). Consistent with our p24 staining results, we found that the largest amount of HIV-1 gag DNA was present in the most recently activated T cells that had not undergone cell division but produced cytokines (Fig. 6 A). The second largest gag DNA content was found in dividing and cytokine-producing T cells. The lowest gag DNA contents were detected in undivided noncytokine-producing cells, indicating that T cells that had not been activated by DCs were less likely to become productively infected by HIV-1. The same pattern was observed using each of the DC subsets exposed to either HIV-1 BaL or HIV-1 IIIB (unpublished data).
The p24 stainings were performed at a time point sufficiently distant from the initial interaction of DCs and T cells. This allows for the possibility that the activation of T cells and any subsequent infection may be temporally and mechanistically distinct. To overcome this problem, we sorted responding and nonresponding T cells very soon (10 h) after the initiation of co-culture with DCs. This provided a very limited time for T cells to receive both activation signals and HIV-1 infection from a DC. Cytokine-producing and noncytokine-producing T cells were sorted and HIV-1-negative strand strong stop DNA (sssDNA), an early product of HIV-1 replication, was assessed by real time PCR (Fig. 6, B and C). Delta count values demonstrating the number of cycles required for the detection of HIV-1 sssDNA were used as an estimate to examine the amount of early reverse transcripts in the various fractions. On average, the responding cytokine-producing T cells contained early reverse transcript levels detectable at five fewer cycles of amplification as compared with noncytokine-producing T cells. This difference translates to -23فfold more early infection processes in responding as compared with nonresponding T cells. This indicates that most infected T cells became activated and infected at the same time, probably when they were still engaged in an immunologic synapse.

HIV-1 is transferred in conjugates of infected DCs and T cells
We have previously shown that in co-cultures of HIV-1infected skin DCs (Langerhans cells) and T cells, the infected p24 ϩ T cells predominantly appear in tight conjugates of T cells and CD1a ϩ Langerhans cells (32). We analyzed whether this observation also held for MDCs and PDCs. We gated on different cell populations within the co-cultures and examined them for p24 versus cytokine expression (Fig. 7, A and  B). Single MDCs or PDCs, expressing the DC-specific markers CD11c or CD123, respectively, but not associated with CFSE-labeled T cells, were analyzed, as were undivided and divided T cells not colocalized with DC markers. A separate

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population of cells that coexpressed DC markers and CFSE that represented cell conjugates was also analyzed. The presence or absence of DCs and T cells within these different cell populations was confirmed by polychromatic flow cytometry examining the expression of CD11c/CD123, CD3, CD4, CD8, CD14, and CD20 (32,33). Conjugate formation between DCs and T cells was observed among both undivided and dividing T cells. Higher proportions of MDCs, as opposed to PDCs, were found in clusters with T cells (Fig. 7, A-D). In addition, DC maturation led to a further increase in cluster formation (unpublished data). Cell conjugates were observed in all cultures whether unexposed or exposed to either HIV-1 BaL or HIV-1 IIIB . The DC-T cell conjugates in co-cultures with either PDCs or MDCs contained significantly higher frequencies of infected p24 ϩ cells as compared with undivided T cells not associated with DCs (n ϭ 10, P ϭ 0.008). T cells that had undergone cell division, and thus been activated by DCs at a prior time point, displayed similar, but most often lower, frequencies of p24 ϩ T cells compared with the T cells in the conjugates. The highest frequencies of cytokine-producing cells were found in the population consisting of cell conjugates as compared with the other three populations (n ϭ 10, P ϭ 0.008). In addition, the proportion of p24 ϩ cells that also produced cytokines was the highest in the conjugates of DCs and T cells as compared with either undivided or divided T cells (Fig. 7, A and B).

DISCUSSION
It is well documented that DCs can efficiently transfer HIV-1 to T cells. However, the exact nature of the T cells that re-  ceive the infection is still unknown. We describe a novel coculture and flow cytometry model system to examine the CD4 ϩ T cells that become infected by HIV-1 from bloodderived subsets of DCs (MDCs and PDCs) during antigen presentation. Our results suggest that DC-mediated HIV-1 infection of CD4 ϩ T cells does not occur randomly. In fact, HIV-1 usurps the antigen presentation and activation functions of DCs to specifically transfer HIV-1 to the T cells that are responding to the antigens that these DCs are presenting. This is possible because HIV-1 infection by itself does not notably diminish the antigen-processing and T cell-activating properties of DCs. HIV-1 produced by DCs is therefore provided an optimally activated CD4 ϩ T cell population in which to establish a productive infection that can lead to the depletion of the specific T cells that are responding to cognate antigens. This would result in a specific loss of important T cell specificities.
It has been suggested that HIV-1-infected DCs will present infectious HIV-1 to HIV-1-specific CD4 ϩ T cells in vivo (1). However, directly testing this in vitro is difficult because it would require the use of DCs and T cells from HIV-1-infected subjects, and few patients have adequate numbers of HIV-1-specific CD4 ϩ T cells to carry out these experiments (34,35). Therefore, we used healthy CMV ϩ subjects with preserved CMV-specific CD4 ϩ T cells to model the transfer of HIV-1 from infected DCs to antigenspecific T cells. In our in vitro system, we showed that HIV-1-infected MDCs and PDCs presenting CMV antigens both activated preexisting autologous CMV-specific memory CD4 ϩ T cells and preferentially transferred the virus to them. This pattern was also seen when using SEB that induced the cross-linking of TCRs as an alternative to using CMV antigens to activate T cells. This mechanism of preferential transfer to antigen-specific T cells may explain why HIV-1-specific T cells in infected individuals more frequently contain HIV-1 than T cells with other TCR specificities (1). It should be noted that the mechanism of infection identified here is not the only route of infection of CD4 ϩ T cells. We have previously shown that naive T cells are infected in vivo (33), and this is unlikely to occur from DCs according to antigen specificities. In addition, we show here that once HIV-1 is transferred from a DC to an antigen-specific T cell, that infection can rapidly spread from T cell to T cell.
Immunological synapses are formed between DCs and T cells during antigen presentation. MHC class II peptidebearing compartments within a DC have been shown to move to the contact point between the DC and the interacting TCR peptide-specific T cell (36,37). HIV-1 may exploit this cellular machinery for transmission from infected DCs to CD4 ϩ T cells. Viruses that spread from DCs to T cells have been shown to be directed to the connection site between the cells (28). DCs replicate HIV-1 poorly as compared with activated CD4 ϩ T cells. Therefore, the efficient transfer of a virus from DCs to T cells may require cell-cell contact (38). We found that HIV-1 p24 was preferentially detected in conjugates of DCs and T cells, suggesting that T cells become activated and infected through cluster formation with infected DCs rather than from a free virus in the culture media secreted by HIV-1-infected DCs. This is also supported by the fact that we found early transcripts of HIV-1 primarily in CMV-specific cytokine-producing T cells, as opposed to nonresponding T cells, only 10 h after co-culture with infected DCs. Still, we have not demonstrated that it is the HIV-1-infected DC that also presents CMV to, and activates, CMV-specific T cells. Therefore, we cannot exclude a scenario in which an uninfected DC presents CMV antigens to a responding CMV-specific T cell that will receive an HIV-1 infection from HIV-1-infected DCs that are in close proximity.
Preferential transfer of the virus to antigen-specific T cells occurred despite the differential susceptibility of DC subsets to R5 (BaL)-and X4 (IIIB)-using virus isolates. We have previously found that TLR7/8 ligation using R-848 led to differentiation of both MDCs and PDCs, which in turn enabled them to activate higher frequencies of antigenspecific T cells (17). We found that R-848-matured MDCs transferred and propagated HIV-1 in T cells more efficiently than immature MDCs. More conjugates were formed between MDCs and T cells when the MDCs were mature, which would facilitate the milieu for viral transfer. Furthermore, the superior activation of T cells induced by mature MDCs can lead to enhanced replication in T cells. Therefore, although mature MDCs are better APCs and are essential for the induction of primary immune responses, they are also better at transferring the virus. This ability of HIV-1 to infect DCs and usurp their functions may accelerate the immune depletion in an HIV-1 infection by specifically depleting responding CD4 ϩ T cells and allowing for an early lacunar defect in the HIV-1-specific repertoire. We did not see an increased transfer of the virus upon maturation of PDCs. Although we have previously found that R-848matured PDCs are better APCs than immature PDCs (17), they may not be superior at transferring the virus to T cells. This may derive from the fact that exposure to either HIV-1 or R-848 will induce IFN-␣ production in PDCs (17)(18)(19), which can prevent viral replication in T cells (39). In addition, PDCs are highly sensitive to long-term culture and stimulation, and may die during the relatively long-term coculture used in our in vitro system. Because of the low frequencies of DCs in vivo and the difficulties in isolating these cells, several different sources for DCs have been used to study the biology of DCs in the context of HIV-1 infection. Numerous in vitro studies have shown that there are at least two pathways by which HIV-1 interacts with DCs (21)(22)(23)(24)(25)(26)(27). These include the conventional CD4-and coreceptor-mediated infection by HIV-1, as well as the binding of the virus via DC-SIGN and other C-type lectins. The mechanism of viral transfer from DCs to T cells may first be dependent on the type of DC that is used.

ARTICLE
Monocyte-derived DCs have been shown to capture HIV-1 without infection and transmit the virus to T cells (23,24,27,29). Viral transmission to T cells from isolated Langerhans cells has been shown to occur in a CD4-dependent manner (22,26,40), but Langerhans cells can also bind HIV-1 in a CD4-independent manner (25). The mechanism of viral transfer may also be dependent on the time of viral exposure (27). It has been shown that the initial phase of HIV-1 transfer from monocyte-derived DCs to T cells is not dependent on a productive infection. The virus captured soon after DC exposure may thereafter be degraded and a productive infection of DCs is then required for viral transfer to T cells to occur at later time points (27). We found that transmission of the virus to T cells by PDCs and MDCs required productive infection by the DCs after 72 h of virus exposure, whereas soon after virus exposure (within 6 h), DCs could pass the virus to T cells without being productively infected. Thus, despite phenotypic and functional differences between MDCs, PDCs, and other DC types, they may all use similar mechanisms to transfer HIV-1 to T cells.
It may be possible to prevent the spread of HIV-1 by blocking conjugate formation between DCs and T cells, and thereby decrease T cell activation and proliferation. On the other hand, as the interaction between DCs and T cells is essential for elicitation of primary and secondary immune responses against pathogens, inhibiting this general interaction would not be without consequences. That said, a better understanding of the process by which HIV-1 targets antigenspecific T cells and that leads to their productive infection will certainly aid in our discovery of new and better approaches to vaccine and therapeutic agents.

MATERIALS AND METHODS
DC isolation. This study was approved by the National Institutes of Health Institutional Review Board. Our sorting procedures for the direct isolation of DC subsets from blood have been described previously (17). In brief, PBMCs were collected from healthy, normal, CMV-seropositive blood donors by automated leukapheresis. Enriched populations of lymphocytes and monocytes were obtained by counterflow centrifugal elutriation. MDCs and PDCs were isolated from elutriated monocytes using magnetic bead isolation, including DC isolation kits (Miltenyi Biotec), followed by sequential separation (AutoMacs; Miltenyi Biotec). The BDCA-4 and the CD1c isolation kits were used for the isolation of PDCs and MDCs, respectively. After this purification, the DC subsets were highly enriched (on average, Ͼ75% of PDCs and Ͼ85% of MDCs) as determined by staining for the lineage markers (CD3, CD14, CD15, CD20, and CD56), HLA-DR, CD123, and CD11c, respectively. Any contaminating cells were mostly monocytes. DCs were cultured in a complete medium of RPMI 1640 (Bio-Whittaker, Inc.) supplemented with 2 mmol/liter L-glutamine, 1% streptomycin and penicillin, and 10% FCS (Invitrogen). To maintain viability, the media for PDCs and MDCs were supplemented with the recombinant human cytokines IL-3 (1 ng/ml; R&D Systems) and GM-CSF (2 ng/ml; Pep-roTech), respectively.

HIV-1 virus growth and preparation.
The CCR5-using HIV-1 BaL isolate and the CXCR4-using HIV-1 IIIB isolate (NIH AIDS Research and Reference Reagent Program) were grown on PHA-(Sigma-Aldrich) and IL-2-(Chiron Corp.) stimulated PBMCs. The p24 Gag level in the cell culture supernatant was monitored by p24 ELISA and the viruses were har-vested at a peak time point. To concentrate the virus and to minimize the presence of bystander activation factors in the supernatant that could influence DC growth, the isolates were concentrated with an ultracentrifugation machine at 30,000 rpm for 70 min at 4ЊC (Sorvall Surespin 630; Kendro). The virus pellet was resuspended in fresh RPMI with 10% FCS to obtain a 5-10-fold concentrated clean virus stock.
Characterization of HIV-1 stocks. The viral titers of the HIV-1 stocks were determined by p24 ELISA (PerkinElmer) according to the manufacturer's instructions. The highly concentrated HIV-1 BaL stock had 10 g/ml HIV-1 p24 Gag and the HIV-1 IIIB stock had 5 g/ml HIV-1 p24. Virus tissue culture ID 50 was determined by a sensitive 14-d endpoint titration assay using PHA-and IL-2-stimulated PBMCs, as previously described (41). The HIV-1 BaL stock had 2.6 ϫ 10 6 tissue culture ID 50 /ml and the HIV-1 IIIB stock 1.75 ϫ 10 6 tissue culture ID 50 /ml.

HIV-1 infection and stimulation of DCs.
The DC populations were cultured at 10 6 cells/ml (with no less than 0.25 ϫ 10 6 cells/tube) for 12 h at 37ЊC in polystyrene round-bottom tubes (Becton Dickinson). HIV-1 BaL or HIV-1 IIIB (normalized by a final concentration of 1 g p24/ml, which translated into a multiplicity of infection of 0.2-0.4) was thereafter added to the DCs, and the cells were cultured for 6 or 72 h similar to as described earlier (16,42). For dose-response comparisons, the virus isolates were used at 0.01-1 g p24/ml. In some experiments, 0.1 M AZT, a nucleoside analogue that inhibits reverse transcription (Sigma-Aldrich), was added to the cultures before addition of the virus isolate. To induce maturation, the DCs were stimulated by the TLR7/8 ligand R-848 (1 g/ml, 4-amino-2-ethoxymethyl-␣, ␣-dimethyl-1H-imidazoquinoline-1-ethanol; GLSynthesis Inc.) as previously described (17). The DCs were exposed to HIV-1 for 72 h and stimulated with R-848 during the final 24 h.

Measurement of IFN-␣ release.
The supernatants from HIV-1exposed and unexposed DCs were harvested and analyzed by ELISA (Biosource International) according to the manufacturer's instructions (17).

Isolation and CFSE labeling of CD4 ؉ T cells.
Enriched populations of autologous negatively selected CD4 ϩ T cells, depleted of CD14 ϩ , CD15 ϩ , CD19 ϩ , CD56 ϩ , CD1c ϩ , BDCA-4 ϩ , and CD8 ϩ cells, were isolated using microbeads (Miltenyi Biotec) on elutriated lymphocytes and sequential separation on AutoMacs. The purity was Ͼ96%. The sorted CD4 ϩ T cells were frozen in 10% DMSO (Fisher Scientific) in FCS and stored at Ϫ85ЊC until use. At the time of co-culture, the CD4 ϩ T cells were thawed in the presence of 0.1 U/ml DNase I (RNase-free; Roche), washed thoroughly, and labeled with 0.25 M CFSE (Molecular Probes) as described previously (31,32). CFSE-labeled cells were washed and resuspended in medium. Cell viability was determined by Trypan blue exclusion before coculture with DCs.
Co-culture of HIV-1-infected DCs with autologous CD4 ؉ T cells. HIV-1-exposed or unexposed DCs were thoroughly washed by 3ϫ repetitive centrifugation in 4 ml of prewarmed media to minimize the trans-fer of residual no-cell-associated virus. The DCs were pulsed with whole CMV antigen lysate. The whole CMV antigen preparation (Microbix Biosystems) was derived from the CMV AD-169 virus strain grown in MRC-5 cells and inactivated by gamma irradiation. The CMV preparation was titrated to find a dose that was not toxic to the cells yet adequate to activate CMV specific memory T cells (10 L/ml). In some experiments, 0.2 g/ml of the TCR superantigen SEB (Sigma-Aldrich) was used as an antigen instead of CMV. The DCs were then co-cultured with sorted autologous CFSElabeled CD4 ϩ T cells in a DC/T cell ratio of 1:10 in complete media in polystyrene round-bottom tubes. The DC-T cell co-cultures were carried for 1-8 d with the addition of 1 g/ml Brefeldin A (Sigma-Aldrich) during the last 12 h of co-culture. In some experiments, 1 M of the protease inhibitor Indinavir (Sigma-Aldrich) was added at initiation or 10 h of co-culture.

Analysis of a DC-mediated transfer of HIV-1 to antigen-specific T cells.
Co-cultures of DCs and T cells were harvested at the indicated time points. Cells were surface stained with anti-CD11c Cy5-PE (for co-cultures including MDCs) or anti-CD123 Cy5-PE (for co-cultures including PDCs). Cells were then fixed and permeabilized in 2ϫ fixation-permeabilization solution (Becton Dickinson) for intracellular staining of HIV-1 p24 and cytokine expression using anti-p24 PE, anti-IFN-␥ APCs, anti-TNF-␣ APCs (BD Biosciences), and anti-IL-2 APCs (Caltag Laboratories). The cells were immediately analyzed on a FACSCalibur flow cytometer to prevent leakage of CFSE dye from the cells. To stringently define the cell types in the co-cultures, a phenotypic analysis was performed in detail with polychromatic flow cytometry using anti-CD11c Cy5-PE/CD123 Cy5-PE, CD3 Cy7-PE, CD4 Cy5.5-PE, CD8 Cy7-APC, CD14 Cy7-APC, and CD20 Cy7-APC as previously described (32,33). Stained cells were analyzed on a modified LSR II (Becton Dickinson). All data were evaluated by FloJo software (Tree Star Inc.).
T cell sorting for the analysis of HIV-1 gag DNA content. DCs were exposed to HIV-1 BaL or HIV-1 IIIB for 72 h and co-cultured with autologous CD4 ϩ T cells as described in Co-culture of...cells section. Co-cultures were harvested at indicated time points and four different fractions of T cells were then sorted on a FACSVantage SE/DiVa (Becton Dickinson). Cells were gated on CD11c Ϫ /CD123 Ϫ CFSE-labeled T cells; subsequently, undivided cytokine-producing, dividing cytokine-producing, undivided noncytokineproducing, and dividing noncytokine-producing T cells were sorted. 15,000-500,000 T cells in each fraction were collected and the pellets were frozen. Viral DNA assessment. The HIV-1 strong stop DNA (an early event of reverse transcription), full-length p17 gag DNA, and human albumin were quantified by real-time PCR on an ABI7900HT (Applied Biosystems) as previously described (1). In brief, the sorted cells were lysed in 0.1 mg/ml proteinase K buffer in Tris-Cl (pH 8.0) at 100,000 cells per 10 l of buffer by incubation at 56ЊC for 1 h and then at 95ЊC for 10 min. The lysate was cleared by centrifugation at 14,000 g for 2 min. 5 l of the lysate was used in a reaction volume of 25 l with 500 nM of primers (AA55, CTGCTA-GAGATTTTCCACACTGACT; M667 ϩ , TAACTAGGGAACCCACT-GCTTAA; gag FWD, GGTGCGAGAGCGTCAGTATTAAG; gag REV, AGCTCCCTGCTTGCCCATA; Albumin Fwd, tgcatgagaaaacgccagtaa; Albumin Rev, atggtcgcctgttcaccaa; Biosource International), 200 nM of probes (strong stop DNA probe, FAM-AGAGTCACACAACAG-ACGGGCACACACT-BHQ1; gag probe, FAM-AAAATTCGGTT-AAGGCCAGGGGGAAAGAA-BHQ1; Albumin probe, FAM-tgacagagtcaccaaatgctgcacagaa-BHQ1; Biosource International), 200 mM dNTPs, 3.5 mM MgCl 2 , and 1.25 mM Blue 636 reference dye with 0.625 U Platinum Taq in the buffer supplied. The conditions were 95ЊC for 15 s and 60ЊC for 1 min for 50 cycles after a 2-min activation at 95ЊC. Quantitation was performed by running standard curves for p17 gag and albumin controls.