Interleukin 7 induces cytokine secretion and tumoricidal activity by human peripheral blood monocytes.

Peripheral blood monocytes can be induced by stimuli such as bacterial lipopolysaccharide (LPS) to secrete an array of cytokines. We have studied the effects of interleukin 7 (IL-7) on human peripheral blood mononuclear cells (PBMC) and found that IL-7 is a relatively potent inducer of IL-6 secretion IL-6 protein levels were determined either by the B9 hybridoma growth factor assay or by enzyme-linked immunosorbent assay, and mRNA for IL-6 was analyzed by Northern hybridization. Detailed examination revealed that, among PBMC, monocytes, rather than lymphocytes, were secreting IL-6 in response to IL-7. In contrast to the low concentrations of IL-7 required to stimulate T cell growth and differentiation (as low as 0.1 ng/ml), relatively high concentrations of IL-7 were necessary to induce IL-6 secretion by monocytes (at least 10 ng/ml). An optimal concentration of IL-7 (100 ng/ml) induced monocytes to secrete 10-fold more IL-6 than an optimal concentration of IL-1 beta (10 ng/ml), and almost as much as LPS. However, significantly more IL-7 than IL-1 beta was required to induce detectable levels of IL-6. The kinetics of IL-6 secretion by monocytes were identical in response to IL-7, IL-1 beta, or LPS, with IL-6 protein detectable in culture supernatants as early as 2 h after the initiation of culture. IL-4 was found to markedly inhibit the ability of IL-7 or LPS to induce IL-6 mRNA and IL-6 secretion. In addition to promoting IL-6 production, IL-7 induced the secretion of immunoreactive IL-1 alpha, IL-1 beta, and tumor necrosis factor alpha (TNF-alpha) by monocytes. IL-7 also induced monocyte/macrophage tumoricidal activity against a human melanoma cell target, an activity that may be related to the secretion of IL-1 alpha, IL-1 beta, and TNF-alpha. Finally, we used a whole blood culture system as a bridge to in vivo analysis to demonstrate that IL-7 induces cytokine secretion in the absence of culture medium, fetal calf serum, and adherence to plastic. Our data suggest that IL-7, in addition to regulating lymphocyte growth and differentiation, has potent effects on cells of the monocytic lineage. Thus, IL-7 may be an important mediator in inflammation and in the macrophage immune response to tumors.

Regulation of cytolytic cell populations from human peripheral blood by B cell stimulatory factor 1 (interleukin 4).

The effects of B cell stimulatory factor 1 (BSF-1) on the generation of human CTL and lymphokine-activated killer (LAK) cells in vitro were investigated. Both L-2 and BSF-1 were potent helper factors for the generation of antigen-specific CTL in MLC; detection of optimal BSF-1-induced CTL activity in this system occurred when BSF-1 was added to cultures after an initial period of activation during which exogenous BSF-1 was not present. In contrast to IL-2, BSF-1 failed to induce an LAK cell population, as detected with Daudi tumor targets, in cultures that had not been allosensitized. Furthermore, when both lymphokines were added together at culture initiation, BSF-1 inhibited the IL-2-driven generation of cytolytic cells. The differential ability of BSF-1 to promote the generation of CTL but not LAK could have important implications for lymphokine-mediated immunotherapy.

Cytokine interactions in human immunodeficiency virus-infected individuals: roles of interleukin (IL)-2, IL-12, and IL-15.

Cytokines have been shown to be powerful regulators of the immune response. In this study, we analyze the effect that the newly recognized cytokine interleukin (IL)-15 has on proliferation and cytokine induction using peripheral blood mononuclear cells (PBMCs) and purified CD4+ T cells from patients infected with human immunodeficiency virus (HIV) who are at various stages in their disease. We observed that IL-15 enhances the proliferative response in a dose-dependent manner from PBMCs of HIV-infected individuals when stimulated by polyclonal mitogen, tetanus toxoid, or HIV-specific antigen. The effects of exogenous IL-15 are substantially diminished by adding a neutralizing antibody to the beta chain of the IL-2 receptor. Moreover, the ability of IL-15 to increase proliferation is enhanced by the presence of endogenous IL-2 produced in the cultures. The effect that exogenous IL-15 had on IL-2, IL-4, and interferon (IFN)-gamma induction from PBMC's or CD4+ T cells in response to mitogen or tetanus toxoid was also examined. This was compared to the effect that exogenous IL-2 and IL-12 had under the same conditions. Addition of IL-2 or IL-15 to short-term in vitro cultures of either PBMCs or CD4+ T cells had little effect on IL-2, IL-4, or IFN-gamma production. By contrast, IL-12 caused substantial enhancement of both IL-2 and IFN-gamma production from these cultures. The role that endogenous cytokines have on IFN-gamma induction was also studied. Addition of a neutralizing antibody to the alpha chain of the IL-2 receptor or IL-12 to antigen stimulated cultures caused a striking decrease in IFN-gamma production. Neutralization of endogenous IL-15 also resulted in diminished IFN-gamma production from cultures stimulated with mitogen. IL-4 and IFN-gamma protein production by PBMCs and CD4+ T cells stimulated with mitogen was assessed to see if we could detect a specific bias of cytokine production. Small amounts of IL-4 were detected from CD4+ T cells but not PBMCs from most individuals tested. IFN-gamma and IL-2, however, were also produced from these same cultures. These results further elucidate the mechanism of cytokine regulation in HIV-infected individuals, and they provide evidence that IL-15 may be a useful immune modulator.

The effect of in vivo IL-7 deprivation on T cell maturation.

A number of previous studies have suggested a key role for interleukin 7 (IL-7) in the maturation of T lymphocytes. To better assess the function of IL-7 in lymphopoiesis, we have deprived mice of IL-7 in vivo by long-term administration of a neutralizing anti-IL-7 antibody. In a previous report (Grabstein, K. H., T. J. Waldschmidt, F. D. Finkelman, B. W. Hess, A. R. Alpert, N. E. Boiani, A. E. Namen, and P. J. Morrissey. 1993. J. Exp. Med. 178:257-264), we used this system to demonstrate the critical role of IL-7 in B cell maturation. After a brief period of anti-IL-7 treatment, most of the pro-B cells and all of the pre-B and immature B cells were depleted from the bone marrow. In the present report, we have injected anti-IL-7 antibody for periods of up to 12 wk to determine the effect of in vivo IL-7 deprivation on the thymus. The results demonstrate a > 99% reduction in thymic cellularity after extended periods of antibody administration. Examination of thymic CD4- and CD8- defined subsets revealed that, on a proportional basis, the CD4+, CD8+ subset was most depleted, the CD4 and CD8 single positive cells remained essentially unchanged, and the CD4-, CD8- compartment actually increased to approximately 50% of the thymus. Further examination of the double negative thymocytes demonstrated that IL-7 deprivation did, indeed, deplete the CD3-, CD4-, CD8- precursors, with expansion of this subset being interupted at the CD44+, CD25+ stage. The proportional increase in the CD4-, CD8- compartment was found to be due to an accumulation of CD3+, T cell receptor alpha, beta + double negative T cells. Additional analysis revealed that anti-IL-7 treatment suppressed the audition/selection process of T cells, as shown by a significant reduction of single positive cells expressing CD69 and heat stable antigen. Finally, the effects of IL-7 deprivation on the thymus were found to be reversible, with a normal pattern of thymic subsets returning 4 wk after cessation of treatment. The present results thus indicate a central role for IL-7 in the maturation of thymic-derived T cells.

Interleukin 10 and interferon gamma regulation of experimental Trypanosoma cruzi infection.

Studies were undertaken to determine whether interleukin 10, (IL-10) a cytokine shown to inhibit interferon gamma (IFN-gamma) production, was involved in Trypanosoma cruzi infections in mice. Exogenous IFN-gamma protects mice from fatal infection with T. cruzi. Furthermore, resistant B6D2 mice developed fatal T. cruzi infections when treated with neutralizing anti-IFN-gamma monoclonal antibody (mAb). Thus, endogenous as well as exogenous IFN-gamma is important in mediating resistance to this parasite. Because both T. cruzi-susceptible (B6) and -resistant (B6D2) mouse strains produced IFN-gamma during acute infection, we looked for the concomitant production of mediators that could interfere with IFN-gamma-mediated resistance to T. cruzi. We found that IL-10-specific mRNA was produced in the spleens of mice with acute T. cruzi infections. In addition, spleen cell culture supernatants from infected B6 mice, and to a lesser extent B6D2 mice, elaborated an inhibitor(s) of IFN-gamma production. This inhibitor(s) was neutralized by anti-IL-10 mAb. These experiments demonstrated the production of biologically active IL-10 during T. cruzi infection. In further studies in vitro, it was shown that IL-10 blocked the ability of IFN-gamma to inhibit the intracellular replication of T. cruzi in mouse peritoneal macrophages. Thus, in addition to its known ability to inhibit the production of IFN-gamma, IL-10 (cytokine synthesis inhibitory factor), may also inhibit the effects of IFN-gamma. These experiments demonstrate that IL-10 is produced during infection with a protozoan parasite and suggest a regulatory role for this cytokine in the mediation of susceptibility to acute disease.

Recombinant granulocyte/macrophage colony-stimulating factor activates macrophages to inhibit Trypanosoma cruzi and release hydrogen peroxide. Comparison with interferon gamma.

Recombinant granulocyte/macrophage colony-stimulating factors (rGM-CSF) of mouse and human origins activated macrophages of the homologous species to inhibit the replication of the protozoan parasite T. cruzi. Activation could be induced with 10-100 ng/ml of rMu-GM-CSF, whether it was added before or after uptake of the parasite, in either adherent or suspension cultures. However, the degree of inhibition of parasite replication after exposure to rMu-GM-CSF was not as great as after treatment with rMu-IFN-gamma, and much more rMu-GM-CSF than rMu-IFN-gamma was required to achieve an equivalent antimicrobial effect. These results were mirrored by effects of the cytokines on enhancement of H2O2-releasing capacity in resident mouse peritoneal macrophages. In the latter tests, rMu-IFN-gamma and rHu-TNF-alpha afforded a 44-51-fold enhancement over the untreated control, with a 50% effective concentration (EC50) for rMu-IFN-gamma of approximately 0.05 ng/ml. Using rMu-GM-CSF or rM-CSF, enhancement of H2O2-releasing capacity was 14-15-fold over control, with EC50s of 1 and 14 ng/ml, respectively. However, peak enhancement of macrophage H2O2-releasing capacity was seen at least 24 h earlier with rMu-GM-CSF or rHu-M-CSF than with r-Mu-IFN-gamma or rHu-TNF-alpha. Thus, rMu-GM-CSF and rHu-GM-CSF displayed clear-cut macrophage-activating activity in vitro, but rMu-GM-CSF was less potent and less effective than rMu-IFN-gamma in the tests used.

Characterization of human CD8+ T cells reactive with Mycobacterium tuberculosis-infected antigen-presenting cells.

Previous studies in murine models, including those using the beta2 microglobulin knockout mouse, have suggested an important role for CD8+ T cells in host defense to Mycobacterium tuberculosis (Mtb). At present, little is understood about these cells in the human immune response to tuberculosis. This report demonstrates the existence of human Mtb-reactive CD8+ T cells. These cells are present preferentially in persons infected with Mtb and produce interferon gamma in response to stimulation with Mtb-infected target cells. Recognition of Mtb-infected cells by these CD8+ T cells is restricted neither by the major histocompatibility complex (MHC) class I A, B, or C alleles nor by CD1, although it is inhibited by anti-MHC class I antibody. The Mtb-specific CD8+ T cells recognize an antigen which is generated in the proteasome, but which does not require transport through the Golgi-ER. The data suggest the possible use of nonpolymorphic MHC class Ib antigen presenting structures other than CD1.

Inhibition of murine B and T lymphopoiesis in vivo by an anti-interleukin 7 monoclonal antibody.

The effects of interleukin 7 (IL-7) on the growth and differentiation of murine B cell progenitors has been well characterized using in vitro culture methods. We have investigated the role of IL-7 in vivo using a monoclonal antibody that neutralizes IL-7. We find that treatment of mice with this antibody completely inhibits the development of B cell progenitors from the pro-B cell stage forward. We also provide evidence that all peripheral B cells, including those of the B-1 and conventional lineages, are derived from IL-7-dependent precursors. The results are consistent with the rapid turnover of B cell progenitors in the marrow, but a slow turnover of mature B cells in the periphery. In addition to effects on B cell development, anti-IL-7 treatment substantially reduced thymus cellularity, affecting all major thymic subpopulations.

Human interleukin 4 receptor confers biological responsiveness and defines a novel receptor superfamily.

IL-4, a pleiotropic cytokine produced by T lymphocytes, plays an important role in immune responsiveness by regulating proliferation and differentiation of a variety of lymphoid and myeloid cells via binding to high affinity receptors. In this report we describe the isolation and functional expression of a human IL-4-R cDNA. When transfected into COS-7 cells, the cDNA encodes a 140-kD cell-surface protein. After transfection into a murine T cell line, the cDNA encodes a protein that binds human IL-4 with high affinity and can confer responsiveness to human IL-4. The predicted extracellular domain of the IL-4-R exhibits significant amino acid sequence homology with the beta subunit of the IL-2-R (p75), and the receptors for IL-6, erythropoietin, and prolactin. These receptors comprise a novel superfamily with extracellular domains characterized by four conserved cysteine residues and a double tryptophan-serine (WSXWS) motif located proximal to the transmembrane region.

Recombinant interleukin 7, pre-B cell growth factor, has costimulatory activity on purified mature T cells.

The activation of highly purified murine peripheral T cells in vitro by Con A is dependent on a co-stimulatory signal that is not IL-1 or IL-2. Previous evidence has demonstrated that the recently defined lymphokine IL-6 could provide costimulatory activity for purified T cells cultured with Con A. In this report we demonstrate that IL-7 also has potent co-stimulatory activity for purified murine T cells, as well as its previously described ability to support the growth of pre-B cells in Witte-Whitlock cultures. When rIL-7 was added to cultures of purified T cells together with Con A, it induced the expression of IL-2 receptors, IL-2 production, and consequently proliferation. In addition, IL-7 exhibited the same magnitude of activity in this assay as IL-6. Also, anti-IL-6 antibody which inhibited the IL-6-induced response had no effect on the IL-7 response. Thus, IL-7 does not act by inducing IL-6. These results demonstrate that IL-7, a potent growth stimulus for pre-B cells, also has a role in T cell activation.

Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice.

Interleukin 7 (IL-7) stimulates the proliferation of B cell progenitors, thymocytes, and mature T cells through an interaction with a high affinity receptor (IL-7R) belonging to the hematopoietin receptor superfamily. We have further addressed the role of IL-7 and its receptor during B and T cell development by generating mice genetically deficient in IL-7R. Mutant mice display a profound reduction in thymic and peripheral lymphoid cellularity. Analyses of lymphoid progenitor populations in IL-7R-deficient mice define precisely those developmental stages affected by the mutation and reveal a critical role for IL-7R during early lymphoid development. Significantly, these studies indicate that the phase of thymocyte expansion occurring before the onset of T cell receptor gene rearrangement is critically dependent upon, and mediated by the high affinity receptor for IL-7.

Induction of macrophage tumoricidal activity by granulocyte-macrophage colony-stimulating factor.

Monocytes are a subpopulation of peripheral blood leukocytes, which when appropriately activated by the regulatory hormones of the immune system, are capable of becoming macrophages--potent effector cells for immune response to tumors and parasites. A complementary DNA for the T lymphocyte-derived lymphokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), has been cloned, and recombinant GM-CSF protein has been expressed in yeast and purified to homogeneity. This purified human recombinant GM-CSF stimulated peripheral blood monocytes in vitro to become cytotoxic for the malignant melanoma cell line A375. Another T cell-derived lymphokine, gamma-interferon (IFN-gamma), also stimulated peripheral blood monocytes to become tumoricidal against this malignant cell line. When IFN-gamma activates monocytes to become tumoricidal, additional stimulation by exogenously added lipopolysaccharide is required. No such exogenous signals were required for the activation of monocytes by GM-CSF.

Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor.

A cytokine was identified that stimulated the proliferation of T lymphocytes, and a complementary DNA clone encoding this new T cell growth factor was isolated. The cytokine, designated interleukin-15 (IL-15), is produced by a wide variety of cells and tissues and shares many biological properties with IL-2. Monoclonal antibodies to the beta chain of the IL-2 receptor inhibited the biological activity of IL-15, and IL-15 competed for binding with IL-2, indicating that IL-15 uses components of the IL-2 receptor.

In vitro responses to Leishmania antigens by lymphocytes from patients with leishmaniasis or Chagas' disease.

T cell responses are correlated with recovery from and resistance to leishmaniasis. Antigens of Leishmania chagasi were evaluated by determining their ability to elicit in vitro proliferation and cytokine production in peripheral blood lymphocytes and in T cell lines and clones from patients with histories of leishmaniasis or Chagas' disease. Antigens tested were selected by their reactivity with patient antibodies. Several of the antigens induced proliferative responses in peripheral blood lymphocytes from patients recovered from visceral or cutaneous leishmaniasis or with chronic Chagas' disease. Two purified glycoproteins, 30 and 42 kD, were consistently among the most effective in eliciting high proliferative responses and IL-2 production. Lymphocytes from a recovered visceral leishmaniasis patient were used to produce T cell lines against either the 30- or 42-kD antigen. Each of the lines responded to both of these antigens as well as to crude leishmania lysate. CD4+ T cell clones specific for either or both of these antigens were also isolated from a visceral leishmaniasis patient. In contrast, rabbit antisera produced against these two antigens were not crossreactive. Both antigens were effective in inducing the production of IFN-gamma from T cell lines from both leishmaniasis and Chagas' disease patients. These studies demonstrate the potential for defining parasite antigens with broad immunostimulatory capabilities.

Stage-specific transformation of murine B lineage cells by ras and myc.

The retroviral vector delta RM coexpresses the v-Ha-ras and v-mycMC29 oncogenes, under the transcriptional control of the retroviral long terminal repeat and an internal SV40 promoter respectively. In this report, the transforming activity of the delta RM virus on murine pre-B cells has been compared and contrasted with its activity on mature splenic B cells. Infection of primary bone marrow cells, followed by growth in the Whitlock-Witte culture system, resulted in the rapid outgrowth of transformed pre-B cells. These cells grew to high saturation densities and could give rise to immortal, interleukin-7-independent progeny that were able to grow independently of stromal elements. In contrast, infection of mature B cells purified from murine spleen resulted in only a transient increase in proliferation, and no immortal B cell lines were obtained. This inability of delta RM to transform mature B lymphocytes was not due to a low infection frequency, since parallel experiments with ecotropic retroviruses conferring drug resistance showed that the mature B cells were readily infectable. Moreover, Northern analysis showed that the delta RM-infected mature B cells expressed ras and myc mRNAs to higher levels than the delta RM transformed pre-B cells. Thus, coexpression of ras and myc resulted in the transformation of primary pre-B cells but not of the mature B cells. The potential explanations for the stage-specific transforming activity of the delta RM retrovirus are discussed.

Generation of immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian cancer using a peptide-based vaccine.

HER-2/neu is a "self" tumor antigen that is overexpressed in 15-30% of human adenocarcinomas. Vaccine strategies directed against HER-2/neu and other self tumor antigens require development of methods to overcome immune tolerance to self-proteins. In rats, rat neu peptide vaccines have been shown to be an effective way of circumventing tolerance to rat neu protein and generating rat neu-specific immunity. The present report validates that a similar peptide-based vaccine formulation is effective for inducing T-cell immunity to HER-2/neu protein in humans with breast and ovarian cancer. The vaccine formulation included groups of peptides derived from the HER-2/neu extracellular domain (ECD) or intracellular domain (ICD) mixed with granulocyte macrophage colony stimulating factor as an adjuvant. These peptides were 15-18 amino acids in length and designed to elicit a CD4 T helper-specific immune response. Patients underwent intradermal immunization once a month for a total of two to six immunizations. To date, all of the patients immunized with HER-2/neu peptides developed HER-2/neu peptide-specific T-cell responses. The majority of patients (six of eight) also developed HER-2/neu protein-specific responses. Responses to HER-2/neu protein occurred with epitope spreading. Immune T cells elicited by vaccination were shown to migrate outside the peripheral circulation by virtue of generating delayed type hypersensitivity responses distant from the vaccine site, which indicated the potential ability to traffic to the site of tumor. The use of peptide-based vaccines may be a simple, yet effective, vaccine strategy for immunizing humans to oncogenic self-proteins.

Proliferative and cytokine responses to class II HER-2/neu-associated peptides in breast cancer patients.

Previous studies have characterized the reactivity of CD8+ CTLs with ovarian and breast cancer. There is little information about the antigens and epitopes recognized by CD4+ T cells in these patients. In this study, we analyzed the ability of T cells from peripheral blood mononuclear cells of breast cancer patients to recognize HER-2/neu (HER-2) peptides. We found that 13 of 18 patients responded by proliferation to at least one of the HER-2 peptides tested. Of these peptides, one designated G89 (HER-2: 777-789) was recognized by T cells from 10 patients. Seven of nine responding patients were HLA-DR4+, suggesting that this peptide is recognized preferentially in association with HLA-DR4. Analysis of the specificity and restriction of the cytokine responses to G89 by G89-stimulated T cells revealed that these cells secreted significantly higher levels of IFN-gamma than interleukin 4 and interleukin 10, suggesting priming for a Th0-T helper 1 response. The same pattern of cytokine responses was observed to the intracellular domain of HER-2 protein, suggesting that G89-stimulated T cells recognized epitopes of the HER-2 protein in association with HLA-DR4. Because HLA-DR4 is present in 25% of humans, characterization of MHC class II-restricted epitopes inducing Th0-T helper 1 responses may provide a basis for the development of multivalent HER-2-based vaccines against breast and ovarian cancer.

IL-15, a novel T cell growth factor that shares activities and receptor components with IL-2.

Interleukin 15 is a newly discovered cytokine that shares biological activities with IL-2 and, like IL-2, is a member of the four-helix bundle cytokine family. We have shown that IL-15 shares components of the receptor for IL-2: the alpha chain of the IL-2R is not required, but both the beta and gamma chains are needed for IL-15 mediated bioactivities. A defect in IL-15 signaling may therefore contribute to the phenotype of X-linked severe combined immunodeficiency in humans, resulting from mutations in the common gamma chain. Differential ability of cells to bind and respond to IL-2 and IL-15 suggested the existence of an additional IL-15 specific receptor component. We identified an IL-15 specific binding protein (IL-15R alpha) on a murine T cell and isolated the corresponding cDNA. The IL-15R alpha is not a member of the hematopoietin receptor superfamily, but is structurally related to the alpha chain of the IL-2R. Differences in the expression pattern of IL-15 and its receptor compared to the IL-2 system suggest unique in vivo roles for IL-15.

Interleukin-15: a novel anabolic cytokine for skeletal muscle.

Interleukin-15 (IL-15) is a recently discovered growth factor which is highly expressed in skeletal muscle. In order to determine a functional role for IL-15 in skeletal myogenesis, the effects of IL-15 on myoblast proliferation and muscle-specific myosin heavy chain (MHC) expression were analyzed using the mouse C2 skeletal myogenic cell line and primary fetal bovine skeletal myogenic cultures. IL-15 had no effect on [3H]thymidine incorporation, nor on the rate of myoblast differentiation, assessed by anti-MHC immunocytochemical staining, in either type of culture. However, Western blot analyses revealed that IL-15 used at concentrations of 10 or 100 ng/ml increased MHC accumulation five-fold in C2 myoblast cultures and 2.5-fold in primary bovine myogenic cultures. Moreover, C2 myotubes formed in the presence of IL-15 appeared larger than controls. These findings indicate IL-15 can stimulate differentiated myocytes and muscle fibers to accumulate increased amounts of contractile proteins. Well-fused primary bovine myogenic cultures treated with the mitotic inhibitor aphidicolin, then administered IL-15 and/or the anabolic growth factor insulin-like growth factor-I (IGF-I), were analyzed for MHC accumulation using Western blots. IL-15 used at 10 ng/ml doubled MHC accumulation and was as effective as IGF-I used at 10 or 100 ng/ml. IL-15 and IGF-I used together increased MHC accumulation close to five-fold, indicating these two factors can act additively on muscle fibers. These findings indicate IL-15 affects parameters associated with skeletal muscle fiber hypertrophy, and suggest that IL-15 may be a novel anabolic agent to increase skeletal muscle mass.

Efficient gene transfer and expression in primary B lymphocytes.

In this study we have used a panel of vectors expressing the chloramphenicol acetyltransferase (CAT) reporter gene under the control of different regulatory elements to optimize gene transfer and expression in primary B lymphocytes. The Moloney murine leukemia virus long terminal repeat (MoMLV LTR) and the SV40 early region promoters, while functional in transfected plasmacytoma cell lines, did not give rise to detectable CAT activity following transfection into primary activated mouse or human B lymphocytes. In contrast, the human cytomegalovirus immediate-early (HCMV-IE) enhancer/promoter functioned in both established and primary B cells. The highest expression levels in the primary cells were obtained with vectors containing the Adenovirus 2 major late promoter or the HCMV-IE enhancer/promoter in combination with the Adenovirus 2 tripartite leader and VA genes. These latter expression cassettes were placed in a retroviral vector with the aim of combining their capacity for high-level gene expression with the efficient stable gene transfer afforded by retroviral infection. Several retroviral constructs were made, some of which were able to generate high virus titers. However all of these underwent deletions during the process of retroviral infection, as judged by Southern analysis of infected cells, indicating that they were not optimal gene transfer vectors. The HCMV enhancer/promoter, which was the most active of the other expression cassettes tested in the primary B cells, was inserted into a retroviral vector which also expressed the hph gene under the transcriptional control of the retroviral LTR. This vector did not undergo rearrangement during the process of retroviral infection, as judged by Southern analysis. The CAT gene was inserted downstream of the HCMV promoter in this vector, and a high-titer retroviral stock was generated. Primary B lymphocytes infected with this vector gave high levels of CAT activity, under conditions in which parallel experiments with the hph drug resistance marker showed that one in 20 of the cells were infected. These experiments demonstrate efficient gene transfer and expression in primary B lymphocytes in vitro.