Tolerance and withdrawal of immunosuppressive drugs in patients given kidney and hematopoietic cell transplants.

Sixteen patients conditioned with total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) were given kidney transplants and an injection of CD34+ hematopoietic progenitor cells and T cells from HLA-matched donors in a tolerance induction protocol. Blood cell monitoring included changes in chimerism, balance of T-cell subsets and responses to donor alloantigens. Fifteen patients developed multilineage chimerism without graft-versus-host disease (GVHD), and eight with chimerism for at least 6 months were withdrawn from antirejection medications for 1-3 years (mean, 28 months) without subsequent rejection episodes. Four chimeric patients have just completed or are in the midst of drug withdrawal, and four patients were not withdrawn due to return of underlying disease or rejection episodes. Blood cells from all patients showed early high ratios of CD4+CD25+ regulatory T cells and NKT cells versus conventional naive CD4+ T cells, and those off drugs showed specific unresponsiveness to donor alloantigens. In conclusion, TLI and ATG promoted the development of persistent chimerism and tolerance in a cohort of patients given kidney transplants and hematopoietic donor cell infusions. All 16 patients had excellent graft function at the last observation point with or without maintenance drugs.

Dendritic cell-based xenoantigen vaccination for prostate cancer immunotherapy.

Many tumor-associated Ags represent tissue differentiation Ags that are poorly immunogenic. Their weak immunogenicity may be due to immune tolerance to self-Ags. Prostatic acid phosphatase (PAP) is just such an Ag that is expressed by both normal and malignant prostate tissue. We have previously demonstrated that PAP can be immunogenic in a rodent model. However, generation of prostate-specific autoimmunity was seen only when a xenogeneic homolog of PAP was used as the immunogen. To explore the potential role of xenoantigen immunization in cancer patients, we performed a phase I clinical trial using dendritic cells pulsed with recombinant mouse PAP as a tumor vaccine. Twenty-one patients with metastatic prostate cancer received two monthly vaccinations of xenoantigen-loaded dendritic cells with minimal treatment-associated side effects. All patients developed T cell immunity to mouse PAP following immunization. Eleven of the 21 patients also developed T cell proliferative responses to the homologous self-Ag. These responses were associated with Ag-specific IFN-gamma and/or TNF-alpha secretion, but not IL-4, consistent with induction of Th1 immunity. Finally, 6 of 21 patients had clinical stabilization of their previously progressing prostate cancer. All six of these patients developed T cell immunity to human PAP following vaccination. These results demonstrate that xenoantigen immunization can break tolerance to a self-Ag in humans, resulting in a clinically significant antitumor effect.

Idiotype vaccination following ABMT can stimulate specific anti-idiotype immune responses in patients with B-cell lymphoma.

Vaccination with the idiotype (Id) protein derived from B-cell malignancies can produce Id-specific immune responses that correlate with improved remission duration and survival rates in patients with follicular non-Hodgkin's lymphoma (NHL). A state of minimal or no residual disease correlates strongly with the laboratory detection of a cellular or humoral immune response. High-dose cytotoxic therapy (HDCT) with autologous stem cell support (autologous bone marrow transplantation [ABMT]) can provide profound cytoreduction of B-cell NHL, but the potential immune suppression associated with myeloablative therapy may compromise a patient's ability to mount a specific immune response. To determine whether patients with NHL could mount detectable immuneresponses following ABMT, Id vaccines were administered at 2 to 12 months following myeloablative therapy to a series of patients with relapsed or resistant B-cell NHL. Two different vaccination strategies produced robust immune responses against KLH in all patients, supporting the capacity of the reconstituted immune system following HDCT to react against a strong antigen. Combining the results from both vaccination strategies, 10 of 12 patients mounted Id-specific humoral or cellular responses. Vaccinations were consistently well tolerated. Of the 12 patients, 7 have experienced prolonged remissions with a follow-up from HDCT ranging from 3 to more than 11 years. Our experience serves to document the ability of the recovering immune system to react against both self and xenotypic antigens and supports the feasibility and safety of antigen-specific vaccination following myeloablative therapy in patients with B-cell NHL.

Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy.

Most tumor-associated antigens represent self-proteins and as a result are poorly immunogenic due to immune tolerance. Here we show that tolerance to carcinoembryonic antigen (CEA), which is overexpressed by the majority of lethal malignancies, can be reversed by immunization with a CEA-derived peptide. This peptide was altered to make it a more potent T cell antigen and loaded onto dendritic cells (DCs) for delivery as a cellular vaccine. Although DCs are rare in the blood, we found that treatment of advanced cancer patients with Flt3 ligand, a hematopoietic growth factor, expanded DCs 20-fold in vivo. Immunization with these antigen-loaded DCs induced CD8 cytotoxic T lymphocytes that recognized tumor cells expressing endogenous CEA. Staining with peptide-MHC tetramers demonstrated the expansion of CD8 T cells that recognize both the native and altered epitopes and possess an effector cytotoxic T lymphocyte phenotype (CD45RA(+)CD27(-)CCR7(-)). After vaccination, two of 12 patients experienced dramatic tumor regression, one patient had a mixed response, and two had stable disease. Clinical response correlated with the expansion of CD8 tetramer(+) T cells, confirming the role of CD8 T cells in this treatment strategy.

Dendritic cells injected via different routes induce immunity in cancer patients.

Dendritic cells (DC) represent potent APCs that are capable of generating tumor-specific immunity. We performed a pilot clinical trial using Ag-pulsed DC as a tumor vaccine. Twenty-one patients with metastatic prostate cancer received two monthly injections of DC enriched and activated from their PBMC. DC were cocultured ex vivo with recombinant mouse prostatic acid phosphatase as the target neoantigen. Following enrichment, DC developed an activated phenotype with up-regulation of CD80, CD86, and CD83 expression. During culture, the DC maintained their levels of various adhesion molecules, including CD44, LFA-1, cutaneous lymphocyte-associated Ag, and CD49d, up-regulated CCR7, but lost CD62 ligand and CCR5. In the absence of CD62 ligand, such cells would not be expected to prime T cells efficiently if administered i.v. due to their inability to access lymphoid tissue via high endothelial venules. To assess this possibility, three patient cohorts were immunized with Ag-pulsed DC by i.v., intradermal (i.d.), or intralymphatic (i.l.) injection. All patients developed Ag-specific T cell immune responses following immunization, regardless of route. Induction of IFN-gamma production, however, was seen only with i.d. and i.l. routes of administration, and no IL-4 responses were seen regardless of route, consistent with the induction of Th1-type immunity. Five of nine patients who were immunized by the i.v. route developed Ag-specific Abs compared with one of six for i.d. and two of six for i.l. routes. These results suggest that while activated DC can prime T cell immunity regardless of route, the quality of this response and induction of Ag-specific Abs may be affected by the route of administration.

Idiotype vaccination using dendritic cells after autologous peripheral blood progenitor cell transplantation for multiple myeloma.

The idiotype (Id) determinants on the multiple myeloma immunoglobulin can serve as tumor-specific antigens. An anti-Id immune response may stem the growth of the malignant clone. We report on 26 patients treated at our institution with high-dose chemotherapy and peripheral blood progenitor cell transplantation (PBPCT) and vaccinated with the Id protein. The patients received chemotherapy and PBPCT to establish a minimal residual disease state. After high-dose therapy, the patients received a series of monthly immunizations consisting of 2 intravenous infusions of dendritic cells (DCs) pulsed with either Id protein or Id coupled with keyhole limpet hemocyanin (KLH) as an immunogenic carrier protein, followed by subcutaneous boosts of Id-KLH conjugates. DCs were obtained in all patients from a leukapheresis product 3 to 9 months after PBPCT. Patients were observed for toxicity, immune responses, and tumor status. The DC infusions and the administration of Id-KLH boosts were well tolerated, with patients experiencing only minor and transient side effects. Of the patients, 24 of 26 generated a KLH-specific cellular proliferative immune response. Only 4 patients developed an Id-specific proliferative immune response. Three of these immune responders were in complete remission at the time of vaccination. A total of 17 patients are alive at a median follow-up of 30 months after transplantation. Id vaccination with autologous DCs is feasible for myeloma patients after transplantation. Id-specific cellular responses can be induced in patients who are in complete remission. Further studies are needed to increase the rate of anti-Id immune responses in patients who do not achieve complete remission.

Clinical transplantation tolerance twelve years after prospective withdrawal of immunosuppressive drugs: studies of chimerism and anti-donor reactivity.

BACKGROUND: Previous studies showed the feasibility of inducing transplantation tolerance to cadaveric renal allografts in patients given pretransplant total lymphoid irradiation (TLI). Microchimerism has been theorized to be an important or necessary factor in long-term graft acceptance and tolerance in humans. METHODS: A cadaveric renal transplant recipient given pretransplant total lymphoid irradiation and withdrawn from immunosuppressive drugs more than 12 years ago was tested for microchimerism using a sensitive nested polymerase chain reaction technique, and for anti-donor reactivity using the mixed leukocyte reaction and an ELISA screen for anti-HLA antibodies. Donor and recipient were mismatched for all HLA-A, B, and DR antigens. RESULTS: The "tolerant" recipient had good graft function, no detectable donor-type cells in the blood by polymerase chain reaction analysis, vigorous reactivity to donor stimulator cells in the mixed leukocyte reaction, and no detectable serum anti-HLA antibodies. CONCLUSIONS: Operational tolerance to HLA-A, B, and DR mismatched organ allografts can be induced prospectively in humans for at least 12 years after withdrawal of immunosuppressive drugs. The allograft can be maintained in the absence of detectable donor microchimerism and in the presence of anti-donor reactivity in the mixed leukocyte reaction, suggesting that neither chimerism nor clonal deletion or anergy of recipient T cells to alloantigens presented by donor Class II HLA molecules is required for persistence of the tolerant state using this total lymphoid irradiation protocol.

Idiotype vaccination using dendritic cells after autologous peripheral blood stem cell transplantation for multiple myeloma--a feasibility study.

The idiotype (Id) determinant on the multiple myeloma (MM) protein can be regarded as a tumor-specific marker. Immunotherapy directed at the MM Id may stem the progression of this disease. We report here on the first 12 MM patients treated at our institution with high-dose therapy and peripheral blood stem cell transplantation (PBSCT) followed by Id immunizations. MM patients received PBSCT to eradicate the majority of the disease. PBSCT produced a complete response in 2 patients, a partial response in 9 patients and stable disease in 1 patient. Three to 7 months after high-dose therapy, patients received a series of monthly immunizations that consisted of two intravenous infusions of Id-pulsed autologous dendritic cells (DC) followed by five subcutaneous boosts of Id/keyhole limpet hemocyanin (KLH) administered with adjuvant. Between 1 and 11 x 10(6) DC were obtained by leukapheresis in all patients even after PBSCT. The administration of Id-pulsed DC and Id/KLH vaccines were well tolerated with patients experiencing only minor and transient side effects. Two of 12 patients developed an Id-specific, cellular proliferative immune response and one of three patients studied developed a transient but Id-specific cytotoxic T-cell (CTL) response. Eleven of the 12 patients generated strong KLH-specific cellular proliferative immune responses showing the patients' immunocompetence at the time of vaccination. The two patients who developed a cellular Id-specific immune response remain in complete remission. Of the 12 treated patients, 9 are currently alive after autologous transplantation with a minimum follow-up of 16 months, 2 patients died because of recurrent MM and 1 patient succumbed to acute leukemia. These studies show that patients make strong anti-KLH responses despite recent high-dose therapy and that DC-based Id vaccination is feasible after PBSCT and can induce Id-specific T-cell responses. Further vaccine development is necessary to increase the proportion of patients that make Id-specific immune responses. The clinical benefits of Id vaccination in MM remain to be determined.

Comparison of primary sensitization of naive human T cells to varicella-zoster virus peptides by dendritic cells in vitro with responses elicited in vivo by varicella vaccination.

Dendritic cells (DC) are potent APC during primary and secondary immune responses. The first objective of this study was to determine whether human DC mediate in vitro sensitization of naive CD4+ T cells to epitopes of the immediate early 62 (IE62) protein of varicella zoster virus (VZV). The induction of CD4+ T cell proliferative responses to eight synthetic peptides representing amino acid sequences of the VZV IE62 protein was assessed using T cells and DC from VZV-susceptible donors. The second objective was to compare in vitro responses of naive T cells with responses to VZV peptides induced in vivo after immunization with varicella vaccine. T cell proliferation was induced by three peptides, P1, P4, and P7, in 71-100% of the donors tested before and after vaccination using DC as APC. Monocytes were effective APC for VZV peptides only after immunization. Two peptides, P2 and P8, induced naive T cell proliferation less effectively and were also less immunogenic for T cells from vaccinated or naturally immune donors. T cell recognition of specific peptides was concordant between naive, DC-mediated responses, and postvaccine responses using monocytes as APC in 69% of comparisons (p = 0.05; chi2); the predictive value of a positive response to an IE62 peptide before immunization for T cell sensitization in vivo was 82%. These observations indicate that primary T cell responses detected in vitro using DC as APC may be useful to characterize the potential immunogenicity of viral protein epitopes in vivo.

Isolation and utilization of human dendritic cells from peripheral blood to assay an in vitro primary immune response to varicella-zoster virus peptides.

A human dendritic cell-based assay used to monitor a T cell proliferation response to viral peptides in vitro is described. Dendritic cells and autologous CD4+ T cells were isolated from peripheral blood by a series of density-gradient centrifugations or magnetic bead separations (or both). Peptides corresponding to residues of the immediate early protein, IE62, of varicella-zoster virus (VZV) were used as stimulating antigens, and persons with no history of varicella and no humoral or cellular immunity to VZV served as naive donors for the assays. Three VZV-susceptible donors were tested, and all demonstrated an in vitro response to multiple VZV peptides. This assay has potential as a screen to establish the immunogenicity of viral antigens in vitro using T cells from naive donors.

CTL response to Mycobacterium tuberculosis: identification of an immunogenic epitope in the 19-kDa lipoprotein.

The successful resolution of infection with Mycobacterium tuberculosis (M.tb) is believed to involve the induction of CTLs that are capable of killing cells harboring this pathogen, although little information is known about the MHC restriction or fine specificity of such CTLs. In this study, we used knowledge of the HLA-A*0201-binding motif and an immunofluorescence-based peptide-binding assay to screen for potential HLA-A*0201-binding epitopes contained in the 19-kDa lipoprotein of M.tb (M.tb19). CD8+ T cells derived from HLA-A*0201+ patients with active tuberculosis (TB) as well as tuberculin skin test-positive individuals who had no history of TB were used as effector cells to determine whether these epitopes are recognized by in vivo-primed CTLs. An in vitro vaccination system using HLA-A*0201+ dendritic cells (DCs) as APCs was used to determine whether these epitopes can sensitize naive CD8+ T cells in vitro, leading to the generation of Ag-specific CTLs. The results show that an HLA-A*0201-binding peptide comprised of residues 88 to 97 of M.tb19 (P88-97) is recognized by circulating CD8+ CTLs from both healthy tuberculin skin test-positive individuals and patients with active TB but not by tuberculin skin test-negative subjects. Moreover, dendritic cells pulsed with this peptide induced class I MHC-restricted CTLs from the T cells of healthy unsensitized persons. Finally, CTL lines that were specific for P88-97 were shown to lyse autologous monocytes that had been infected acutely with the H37Ra strain of M.tb. These results demonstrate that M.tb19 elicits HLA class I-restricted CTLs in vitro and in vivo that recognize endogenously processed Ag. Epitopes of the type identified here may prove useful in the design of an M.tb vaccine.

A pilot clinical trial of HIV antigen-pulsed allogeneic and autologous dendritic cell therapy in HIV-infected patients.

A pilot study was carried out to assess the safety and antigen-presenting properties of allogeneic or autologous dendritic cells (DCs) in six HLA-A2+, HIV-infected patients. Allogeneic DCs obtained from the peripheral blood of HLA-identical, HIV-seronegative siblings were pulsed with recombinant HIV-1 MN gp160 or synthetic peptides corresponding to HLA-A2-restricted cytotoxic epitopes of envelope, Gag, and Pol proteins. The antigen-pulsed cells were infused intravenously six to nine times at monthly intervals and HIV-specific immune responses were monitored. One allogeneic DC recipient with a CD4+ T cell count of 460/mm3 showed increases in envelope-specific CTL- and lymphocyte-proliferative responses, as well as in IFN-gamma and IL-2 production. Another allogeneic DC recipient with a CD4+ T cell count of 434/mm3 also showed an increase in HIV envelope-specific lymphocyte-proliferative responses. A recipient of autologous DCs with a CD4+ T cell count of 730/mm3 showed an increase in peptide-specific lymphocyte-proliferative responses after three infusions. Three other allogeneic DC recipients with CD4+ T cell counts <410/mm3 did not show increases in their HIV-specific immune responses. No clinically significant adverse effects were noted in this study and CD4+ T cell numbers and plasma HIV-1 RNA detected by RT-PCR of all six patients were stable during the study period. Thus, both allogeneic and autologous DC infusions were well tolerated and in patients with normal or near normal CD4+ T cell counts administration of these antigen-pulsed cells enhanced the immune response to HIV. However, since no effect on viral load was observed there was no evidence that this approach provided clinical benefit.

Generation of primary peptide-specific CD8+ cytotoxic T-lymphocytes in vitro using allogeneic dendritic cells.

Dendritic cells (DC) are potent antigen-presenting cells (APC) capable of inducing strong T-cell-mediated immunity. Infusion of lymphoma-specific antigen-loaded autologous DC has been demonstrated to result in the generation of antigen-specific immunity and reduction in tumor burden in B-cell lymphoma patients. Cellular immunotherapy employing antigen-loaded DC could have a potential therapeutic impact in tumors and viral infections, including HIV infection. However, DC in HIV-infected individuals and breast cancer patients are believed to be functionally defective. Therefore, the potential of using allogeneic DC offers significant implications for DC immunotherapy in AIDS and immunocompromised cancer patients. To explore the potential of allogeneic DC therapy in vivo, we tested the ability of allogeneic DC to generate primary peptide-specific CD8+ cytotoxic T-lymphocyte (CTL) responses in vitro. Our results indicate that DC from HLA class I-matched individuals elicit primary immune responses in vitro using viral peptides as naive antigens. A primary peptide-specific immune response could also be detected even when only one HLA allele (HLA-A*0201) was matched between the allogeneic DC and T-lymphocytes. The ability to generate primary peptide-specific responses in vitro is strongly indicative of the in vivo therapeutic potential of allogeneic DC.

Dendritic cells that process and present nominal antigens to naive T lymphocytes are derived from CD2+ precursors.

Dendritic cells (DC) are potent APC that, in mature form, can be distinguished from other mononuclear cells on the basis of their distinct morphology, absence of lineage markers, and dense expression of MHC and costimulatory molecules. While comparing different DC preparation methods, we observed that DC derived from cultured PBMC that had been depleted of CD2+ cells before culture were functionally distinct from DC derived from PBMC that had not been depleted of CD2+ cells. Thus, both types of DC stimulated allogeneic T cells to proliferate in the MLR, but only DC derived from CD2+ precursors could sensitize naive T cells to soluble Ags such as keyhole limpet hemocyanin and HIV gp160 glycoprotein. Subsequent studies confirmed the existence of CD2+ and CD2- DC precursor populations among HLA-DRbright, lineage-negative PBMC. Immediately after their isolation, these populations were morphologically similar to one another by light and electron microscopy, and neither had substantial Ag-presenting activity. After culture for 24 to 48 h with supernatant from PHA-activated PBMC, both populations developed dendrites, formed clusters with T cells, and stimulated allogeneic T cell responses in the MLR as well as autologous T cell responses to tetanus toxoid, a recall Ag. However, CD2+ DC precursors alone gave rise to APC that presented soluble Ags to naive CD4+ T cells, a property that could be inhibited by Abs to CD4, CD11a, and CD28 on T cells or CD86 on DC. The expression of CD54 and CD86 on CD2+ DC precursors was increased markedly after their culture and differentiation, while the expression of these molecules on CD2- DC precursors was not remarkably changed. These findings reveal the existence of two functionally distinct populations of DC, each derived from a phenotypically distinct precursor present in monocyte-depleted peripheral blood.

Allogeneic dendritic cell induction of HIV-specific cytotoxic T lymphocyte responses from T cells of HIV type 1-infected and uninfected individuals.

The potential benefit of T cell-based vaccination for HIV-1 infection remains to be determined. Cytotoxic T lymphocytes (CTLs) appear to clear substantial populations of HIV-1 virus in vivo, although CTL activity may contribute to the decline in CD4+ T cell count observed in the course of the disease. To investigate further the role of specific CTL responses in the control of HIV-1 replication, we raised primary CTL lines against a panel of conserved HIV-1 epitopes using blood-derived dendritic cells as antigen-presenting cells (APCs). Specific primary human CTL responses were induced against HLA-A*0201-restricted peptides with dendritic cells from HIV-1-seronegative donors. This method of immunization elicited cytotoxic activities capable of recognizing endogenously processed antigen. The CTL induction protocol was extended in order to explore the capacity of HLA-matched allogeneic dendritic cells to evoke novel CTL responses in T cells from an HIV-seropositive asymptomatic individual. Allogeneic peptide-pulsed dendritic cells from a healthy sibling were capable of eliciting a CTL response directed against an HIV epitope (env814: SLLNATDIAV) that was initially not detected in the CTL effector population of the HIV-1-infected patient. The possibility of manipulating CTL specificity directed against multiple conserved HIV-1 epitopes represents a significant step in the evaluation of T cell-based vaccination for treatment of disease.

Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells.

In this pilot study, we investigated the ability of autologous dendritic cells pulsed ex vivo with tumor-specific idiotype protein to stimulate host antitumor immunity when infused as a vaccine. Four patients with follicular B-cell lymphoma received a series of three or four infusions of antigen-pulsed dendritic cells followed, in each instance, by subcutaneous injections of soluble antigen two weeks later. All patients developed measurable antitumor cellular immune responses. In addition, clinical responses have been measured with one patient experiencing complete tumor regression, a second patient having partial tumor regression, and a third patient resolving all evidence of disease as detected by a sensitive tumor-specific molecular analysis.

V7, a novel leukocyte surface protein that participates in T cell activation. I. Tissue distribution and functional studies.

Among a panel of mouse mAbs generated to a human T cell clone, one mAb, V7.1, inhibited T cell activation in the mixed lymphocyte reaction and was studied further. V7.1 reacted strongly with Ag-specific T cell clones, in addition to freshly isolated monocytes and granulocytes. However, the mAb reacted weakly with freshly isolated PBLs (T cells, B cells, and NK cells), T cells stimulated with phytohemagglutinin, or Con A, and did not stain the vast majority of transformed cell lines of hemopoietic origin. Stimulation of T cells with anti-CD3, or the combination of anti-CD3 and PMA, or anti-CD3, PMA and ionomycin, markedly increased V7.1 surface staining. The mAb precipitated a single polypeptide chain of approximately 135 kDa from alloactivated T cells or monocytes, which was reduced to approximately 110 kDa after treatment with N-glycanase. The proliferative response of T cells to allogeneic monocytes or B lymphoblastoid cells was inhibited by V7.1, and inhibition was maximal when the mAb was present at the initiation of culture. V7.1 also exhibited dose-dependent inhibition of the T cell response to immobilized anti-CD3 Ab in the absence of APCs, indicating that the inhibitory effect of this Ab occurs at the T cell level. Expression of CD25 (IL-2R) on anti-CD3-activated T cells and secretion of IL-2 induced with anti-CD3 and PMA were inhibited by V7.1, whereas the Ab had no effect on T cell proliferation induced by PHA or Con A or on T cell-mediated cytotoxicity. These results indicate that V7.1 recognizes a novel leukocyte surface glycoprotein, designated V7, that is up-regulated on Ag but not lectin-activated T cells, and appears to play a role in TCR/CD3-dependent T cell activation. In an accompanying study, the gene encoding the V7 Ag is described and the molecule is shown to be a novel member of the Ig superfamily.

Evidence that T cell activation is required for HIV-1 entry in CD4+ lymphocytes.

The relationship of T cell activation to HIV entry and generation of viral DNA intermediates was studied in freshly isolated CD4+ T lymphocytes. Unstimulated cells exposed to infectious virus for up to 48 h did not synthesize any detectable unintegrated HIV DNA duplex forms or integrated genomic provirus. However, activation of these cells with either PHA or OKT3 (anti-CD3) mAb before viral exposure resulted in the generation of unintegrated HIV DNA after 6 h and integrated copies after 24 h. Cell-to-cell fusion studies showed significantly attenuated fusion between freshly isolated resting T cells and T cells constitutively expressing high levels of HIV envelope glycoprotein (HXB/gpt) compared with T cells first stimulated with either PHA or OKT3 mAb. The baseline fusion observed with resting T cells is believed to be a consequence of allogeneic stimulation by the HXB/gpt cell line. These results provide evidence that HIV entry and HIV envelope-dependent cell-to-cell fusion require T cell activation.