the absence of an opportunistic infection, there is little or no viral replication in the lung even in patients with ad- vanced AIDS (1). TB markedly increases HIV-1 replication and mutation in involved lung segments (2). Macrophages are the major cell type in which HIV-1 replication occurs in patients with opportunistic infections including TB (3). Activation of HIV-1 replication during opportunistic in- fection may underlie the increased mortality observed in patients coinfected with HIV-1 and TB (4). The CCAAT enhancer binding protein (C/EBP ) gene is the predominant C/EBP isoform expressed in alve- olar macrophages (AM) (5). C/EBP has a stimulatory 37-kD isoform and an inhibitory 16-kD isoform. The inhibi- tory isoform is dominant–negative, repressing promoters with C/EBP sites when expressed at 20% of the level of the stimulatory 37-kD isoform (6). Multiple regulators of in- flammation such as TNF- have C/EBP sites in their pro- moters (7). The serum response factor, a global activator of inflammation, is also suppressed by inhibitory C/EBP (8), which leads to the hypothesis that this dominant–negative transcription factor is responsible for maintaining AM in their baseline quiescent state. The C/EBP family of transcription factors is essential for HIV-1 replication in macrophages but not in lymphocytes (9). There are three C/EBP binding sites present in the negative regulatory element (NRE) of the HIV-1 long ter- minal repeat (LTR) (10). AM from normal lung strongly express an inhibitory 16-kD C/EBP transcription factor that represses the HIV-1–LTR activity in model systems (11). AM from lung segments involved with TB lose ex- pression of inhibitory 16-kD C/EBP , which raises the possibility that derepression is needed before the HIV-1 LTR can be maximally stimulated. Activation of the 5 HIV-1–LTR promoter is an essential step in the viral life cycle. The nuclear factor (NF)- B binding site in the HIV-1 LTR is essential for promoter activity and leads to tran- scriptional induction of viral replication in both lympho- cytes and macrophages (12, 13). In vitro infection of macrophages with M. tuberculosis fails to reproduce loss of the inhibitory 16-kD C/EBP isoform, or the increase in HIV-1 replication, observed in involved lungs of AIDS patients with TB (14). Allogeneic lymphocytes are able to increase HIV-1 replication in mac- rophages (15). Further, isolated membranes from activated lymphocytes enhance HIV-1 replication in macrophages (16). Because cell-mediated immunity requires interaction between lymphocytes and macrophages, we hypothesized that activated lymphocytes were essential to reproduce macrophage activation observed in vivo. We found that lymphocyte contact was required to down-regulate inhibi- tory C/EBP , and that soluble factors activated NF- B. Both contact and soluble factors were required for maximal HIV-1–LTR induction. Materials and Methods Study Population. We performed bronchoscopy on two pa- tients with stable HIV infection without pulmonary disease (see Fig. 2, Patients 6 and 7) and 1 HIV-1–infected patient with ac- tive pulmonary TB (see Fig. 2, Patient 5). The TB patient had unilateral segmental infiltrates. Radiographically uninvolved lobes were identified and a separate bronchoalveolar lavage (BAL) was performed and processed from these segments. The BAL protocol was approved by the Human Subjects Review Committees of New York University Medical Center and Bellevue Hospital Center, and was performed as described (2). BAL cells were centrifuged, resuspended in RPMI 1640 (Bio- Whittaker) with 10% FCS (Life Technologies), and allowed to adhere to plastic plates for 3 h. Cells were recovered by gentle scraping with a rubber policeman. AM were 95% pure by mor- phology and nonspecific esterase staining. HIV-1 viral loads were quantitated by RT-PCR assay (AMPLICOR HIV-1 Monitor™ Test; Roche Molecular Systems). Immunohistochemistry. We used autopsy (see Fig. 1, Patients 1, 2, and 4) or transbronchial lung biopsy samples (see Fig. 1, Pa- tient 3) from HIV patients coinfected with TB. Patient 1 was dead of miliary TB, acute renal failure, and pulmonary edema. Patient 2 was dead of miliary TB, and hilar and mediastinum lymph node TB. Specimens were fixed in periodate/lysine/ paraformaldehyde at 4 C for 10 h. Fixed specimens were dehy- drated with sucrose gradient and embedded in Histofine Sim- plestain (Nichirei), frozen in liquid nitrogen, and sectioned (5 m thickness) using a cryostat. Nonspecific staining was blocked with 10 mM Tris, 5 mM EDTA, 0.15 M NaCl, 0.25% gelatin, 0.05% (vol/vol) Tween 20, and pH 8.0, for 30 min at room tem- perature. After washing with TBS, one of the following primary antibodies was applied: anti–C/EBP rabbit and anti–NF- B polyclonal antibody (1:100; Santa Cruz Biotechnology, Inc.) or anti-CD68 monoclonal (1:100; Dako). After incubating for 1 h at room temperature, sections were washed in TBS four times and incubated with peroxidase-labeled anti–rabbit or anti–mouse an- tibody (Histofine Simplestain Max PO; Nichirei) for 30 min at room temperature. Peroxidase activity was detected with diami- nobenzidine (DAB; Sigma-Aldrich). Sections were counter- stained with hematoxylin and dehydrated. Purification and Activation of T Lymphocytes. PBMC were pu- rified over Ficoll-Hypaque (Amersham Pharmacia Biotech) sedi- mentation. T lymphocytes were separated by nylon wool (Rob- bins Scientific Corp.). The purity of T lymphocytes was more than 90% by flow cytometry (FACScan®; Becton Dickinson) us- ing FITC–anti-CD3 antibodies (Becton Dickinson). To obtain CD4 or CD8 T lymphocytes, the Rosettesep (StemCell Tech- nologies Inc.) was used for negative selection. The purity of CD4 or CD8 lymphocytes was more than 95% by flow cy- tometry (FACScan; Becton Dickinson) using PE–anti-CD4 or PE–anti-CD8 antibodies (Becton Dickinson). T lymphocytes were activated by Con A (Amersham Pharmacia Biotech), 2.5 g/ml, or anti–human CD3 antibodies (R&D Systems), 5 g/ ml, for 48 h as described. Blocking of T Lymphocytes and Cross-linking of Antibodies. Where indicated, Con A– (or anti-CD3–) activated T cells were pretreated with culture medium containing anti-CD28 antibodies (25 g/ml), anti-CD40 ligand (CD154) antibodies (25 g/ml), and anti–very late antigen (VLA)-4 (CD49 ) antibodies (25 g/ ml) (R&D Systems) for 60 min at 4 C before coculture with THP-1 cells or AM. For cross-linking assays, protein A/G agarose beads (100 l; Santa Cruz Biotechnology, Inc.) were mixed with anti–B7-1 (CD80) antibodies (25 g/ml), anti–B7-2 (CD86) an- tibodies (25 g/ml), anti-CD40 antibodies (25 g/ml), and anti– vascular cell adhesion molecule (VCAM)-1 (CD106) antibodies (25 g/ml) (R&D Systems), or control goat serum (Santa Cruz 496 Contact and Cytokines Are Needed for Maximal HIV Replication