Considerations for successful cancer immunotherapy in aged hosts.

Immunotherapy is now experiencing unprecedented successes in treating various cancers based on new understandings of cancer immunopathogenesis. Nonetheless, although ageing is the biggest risk factor for cancer, the majority of cancer immunotherapy preclinical studies are conducted in young hosts. This review will explore age-related changes in immunity as they relate to cancer immune surveillance, immunopathogenesis and responses to immunotherapy. Although it is recognized that declining T cell function with age poses a great challenge to developing effective age-related cancer immunotherapies, examples of successful approaches to overcome this hurdle have been developed. Further, it is now recognized that immune functions do not simply decline with age, but rather change in ways than can be detrimental. For example, with age, specific immune cell populations with detrimental functions can become predominant (such as cells producing proinflammatory cytokines), suppressive cells can become more numerous or more suppressive (such as myeloid-derived suppressor cells), drugs can affect aged immune cells distinctly and the aged microenvironment is becoming recognized as a significant barrier to address. Key developments in these and other areas will be surveyed as they relate to cancer immunotherapy in aged hosts, and areas in need of more study will be assessed with some speculations for the future. We propose the term 'age-related immune dysfunction' (ARID) as best representative of age-associated changes in immunity.

Establishment and characterization of a new mantle cell lymphoma cell line, Mino.

Mantle cell lymphoma (MCL) is a distinct type of B-cell non-Hodgkin's lymphoma characterized by cyclin D1 overexpression and the cytogenetic abnormality, the t(11;14)(q13;q32). MCL cell lines have been difficult to establish and in vitro studies of these neoplasms are scarce. We describe the establishment and characteristics of a new MCL cell line, Mino. The cells are large, growing singly and in small clumps in vitro. By flow cytometry, the immunophenotype was compatible with MCL (i.e. CD5+CD20+CD23-FMC7+). Conventional cytogenetics showed hyperdiploidy with multiple complex karyotypic abnormalities, but no evidence of the t(11;14), proven to be present only by fluorescence in situ hybridization and polymerase chain reaction (PCR) methods. Western blots showed expression of cyclin D1 but no detectable cyclin D2 and cyclin D3; the retinoblastoma protein was predominantly phosphorylated. There was expression of tumor suppressor gene products including p53, p16(INK4a), and p21(WAF1). Sequencing of the TP53 gene revealed a mutation (codon 147(valine-->glycine)) in exon 5. Epstein Barr virus was absent. In summary, Mino is a new MCL cell line that may be useful to study the pathogenesis of MCL.

Stromal-derived factor-1 in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells.

Dendritic-cell (DC) trafficking and function in tumors is poorly characterized, with studies confined to myeloid DCs (DC1s). Tumors inhibit DC1 migration and function, likely hindering specific immunity. The role of plasmacytoid DCs (DC2s) in tumor immunity is unknown. We show here that malignant human ovarian epithelial tumor cells express very high levels of stromal-derived factor-1, which induces DC2 precursor (preDC2) chemotaxis and adhesion/transmigration, upregulates preDC2 very late antigen (VLA)-5, and protects preDC2s from tumor macrophage interleukin-10-induced apoptosis, all through CXC chemokine receptor-4. The VLA-5 ligand vascular-cell adhesion molecule-1 mediated preDC2 adhesion/transmigration. Tumor preDC2s induced significant T-cell interleukin-10 unrelated to preDC2 differentiation or activation state, and this contributed to poor T-cell activation. Myeloid precursor DCs (preDC1s) were not detected. Tumors may weaken immunity by attracting preDC2s and protecting them from the harsh microenvironment, and by altering preDC1 distribution.

Reciprocal regulation of plasmacytoid dendritic cells and monocytes during viral infection.

Reciprocal regulation of opposing functions characterizes biological systems. We now show that adenovirus-infected plasmacytoid dendritic cells (PDC) inhibit monocyte to myeloid dendritic cell (MDC) differentiation and function, and that adenovirus-infected monocytes inhibit PDC type I interferon secretion. Adenovirus-infected PDC secreted IFN-alpha, beta and omega in an 86:2:1 ratio. PDC type I interferons inhibited MDC differentiation and function (reduced IL-12 secretion, IFN-gamma induction, MLR and CD40 expression, and increased CD1a(+)CD14(+) cells). Type I interferon receptor blocking antibody reversed all PDC effects, and recombinant IFN-alpha, beta or omega replicated all effects, except reduced CD40. Adenovirus-infected monocytes suppressed PDC type I interferon secretion, which was reversed with anti-IL-10 neutralizing antibodies. Exogenous IL-10 suppressed PDC type I interferon secretion without reducing PDC viability. Therefore, monocyte IL-10 regulates PDC type I interferon secretion, and PDC type I interferons inhibit MDC differentiation and function. Such reciprocal regulation of potentially opposing influences may help modulate anti-pathogen immunity.

Macrophage-derived dendritic cells have strong Th1-polarizing potential mediated by beta-chemokines rather than IL-12.

Monocyte-derived dendritic cells (MDDCs) activate naive T lymphocytes to induce adaptive immunity, effecting Th1 polarization through IL-12. However, little is known about other potential DC Th1 polarizing mechanisms, or how T cell polarization may be affected by DCs differentiating in, or exposed to, a proinflammatory environment. Macrophages (MPhis) are DC precursors abundant in inflamed tissues, lymph nodes, and tumors. Thus we studied the T cell-activating and -polarizing properties of MPhi-derived DCs (PhiDCs). Monocytes were cultured in MPhi-CSF (M-CSF) to produce MPhis, which were then differentiated into DCs following culture with GM-CSF plus IL-4. PhiDCs activated a significant allogeneic MLR and were significantly better than MDDCs in activating T cells with superantigen. Most strikingly, PhiDCs elicited up to 9-fold more IFN-gamma from naive or Ag-specific T cells compared with MDDCs (with equivalent IL-4 secretion), despite producing up to 9-fold less IL-12. Neutralization of MDDC, but not PhiDC IL-12 significantly inhibited T cell IFN-gamma induction. PhiDCs produced up to 12-fold more beta-chemokines (macrophage-inflammatory protein-1alpha, -1beta, and RANTES) than MDDCs. Ab blockade of CCR5, but not CXC chemokine receptor 4, inhibited T cell IFN-gamma induction by PhiDCs significantly greater than by MDDCs. Thus DCs differentiating from MPhis induce T cell IFN-gamma through beta-chemokines with little or no requirement for IL-12. Myeloid DCs arising from distinct precursor cells may have differing properties, including different mechanisms of Th1 polarization. These data are the first reports of IFN-gamma induction through chemokines by DCs.

Maturation of dendritic cells accompanies high-efficiency gene transfer by a CD40-targeted adenoviral vector.

Important therapeutic applications of genetically modified dendritic cells (DC) have been proposed; however, current vector systems have demonstrated only limited gene delivery efficacy to this cell type. By means of bispecific Abs, we have dramatically enhanced gene transfer to monocyte derived DC (MDDC) by retargeting adenoviral (Ad) vectors to a marker expressed on DC, CD40. Adenovirus targeted to CD40 demonstrated dramatic improvements in gene transfer relative to untargeted Ad vectors. Fundamental to the novelty of this system is the capacity of the vector itself to modulate the immunological status of the MDDC. This vector induces DC maturation as demonstrated phenotypically by increased expression of CD83, MHC, and costimulatory molecules, as well as functionally by production of IL-12 and an enhanced allostimulatory capacity in a MLR. In comparing this vector to other Ad-based gene transfer systems, we have illustrated that the features of DC maturation are not a function of the Ad particle, but rather a consequence of targeting to the CD40 marker. This vector approach may thus mediate not only high-efficiency gene delivery but also serve a proactive role in DC activation that could ultimately strengthen the utility of this vector for immunotherapy strategies.

Efficient gene delivery into epstein-barr virus (EBV)-transformed human B cells mediated by replication-defective herpes simplex virus-1 (HSV-1): A gene therapy model for EBV-related B cell malignancy.

Subgroups of the B cell malignancies are known to be associated with Epstein-Barr virus (EBV) infection, especially in immunocompromised patients. These are fatal and refractory to conventional antineoplastic therapy. B cells are usually post-mitotic cells and even mitogen activated or transformed B cells have shown relative resistance against viral mediated gene transfer. To address this issue, we employed a replication-defective herpes simplex virus-1 (HSV-1) to mediate gene transfer into EBV-transformed B cells. The virus expresses the herpes simplex virus thymidine kinase (HSV-TK) and the E. coli lacZ reporter genes and is designated T0Z.1. We used the lymphoblastoid cell line SWEIG as a model for human EBV-related B cell malignancy. This cell line was established by in vitro EBV infection of primary human peripheral blood mononuclear cells. When SWEIG cells were infected with T0Z.1, X-gal staining revealed lacZ expression in more than 20% cells even at multiplicity of infection (MOI) as low as 1 and the expression persisted for at least one week. Ganciclovir (GCV) administration after T0Z.1 infection effectively decreased the number of the infected tumor cells in a dose-responsive manner. Viral toxicity was analyzed by cell proliferation assay (MTS assay) and found to be little even at 10 MOI infection. Three MOI of the virus yielded maximum antineoplastic effect and more than 50% tumor cells were killed by HSV-TK/GCV. These results suggest the potential utility of replication-defective HSV-1 for the treatment of EBV-related B cell malignancies.

Selection and characterization of Toxoplasma gondii mutants resistant to artemisinin.

Toxoplasma gondii infection, like malaria, is sensitive to inhibition by artemisinin (ART). Mechanisms of action for ART in malaria treatment have been proposed, but little is known about its effects in T. gondii infection. To better understand its inhibitory effects on T. gondii, mutants resistant to ART were selected by progressive culture in permissive levels of the drug. Five clonal isolates were established and characterized. The isolates were approximately 65-fold less sensitive to ART than is the parental RH and showed cross-resistance to the ART derivatives dihydroartemisinin and artemether. In addition to ART resistance, 1 clone (C9) formed morphologically unusual parasitophorous vacuoles and another (A2) was avirulent for mice and protected mice from challenge with the wild type. These clonal T. gondii mutant isolates will be useful for the study of not only the mechanism of action of ART but also parasite vacuole biology and virulence factors.

Stimulation of human T lymphocytes obtained from Toxoplasma gondii-seronegative persons by proteins derived from T. gondii.

Toxoplasma gondii antigens are superantigens in mice. To investigate a superantigen effect in humans, lymphocytes from T. gondii-seronegative subjects were studied for proliferation to T. gondii antigens (TA). Marked cellular proliferation, predominantly of CD4+ lymphocytes, was apparent. TA elicited expansions of Vbeta-bearing lymphocytes in all subjects, but different Vbeta-bearing lymphocytes were expanded in different subjects in both CD4+ and CD8+ subpopulations. Cord blood cells also proliferated to TA. Previously fixed antigen-presenting cells were unable to present TA. Thus, T. gondii appears to produce a molecule(s) that induces polyclonal activation of human T cells and requires antigen processing to mediate this effect. That T. gondii does not appear to behave as a superantigen in humans is important in understanding the pathogenesis of T. gondii infection in immunocompromised hosts and in the design of anti-T. gondii vaccines.

Toxoplasma gondii-infected cells are resistant to multiple inducers of apoptosis.

Infection with certain intracellular pathogens, including viruses and bacteria, may induce host cell apoptosis. On the other hand, infection with some viruses inhibits apoptosis. Complex protozoan parasites, including Toxoplasma gondii and members of Plasmodium, Leishmania, and Microsporidia, are also obligate intracellular pathogens, yet relatively little is known regarding their subversion of host cell functions. We now report that cells infected with T. gondii are resistant to multiple inducers of apoptosis, including Fas-dependent and Fas-independent CTL-mediated cytotoxicity, IL-2 deprivation, gamma irradiation, UV irradiation, and the calcium ionophore beauvericin. Inhibition of such a broad array of apoptosis inducers suggests that a mechanism common to many, or perhaps all, apoptotic pathways is involved. The inhibitory activity requires live intracellular parasite and ongoing protein synthesis. Despite T. gondii-mediated inhibition of DNA fragmentation, infected cells can still be lysed by CTL.

Phenotypic knock-out of the latent membrane protein 1 of Epstein-Barr virus by an intracellular single-chain antibody.

Epstein-Barr virus (EBV) causes lymphoproliferative diseases in immunocompromised patients and is associated with endemic Burkitt lymphoma, nasopharyngeal carcinoma and some cases of Hodgkin disease. The latent membrane protein 1 (LMP1) of EBV is a transmembrane protein that is essential for the transformation of B lymphocytes. LMP1-mediated up-regulation of Bcl-2 is thought to be an important element in this process. As an approach to explore novel treatments for EBV-associated lymphomas, we constructed a single-chain antibody (sFv) directed against LMP1 to achieve functional inhibition of this oncoprotein in EBV-transformed B lymphocytes. We demonstrated that intracellular expression of an endoplasmic reticulum (ER)-targeted form of this sFv markedly reduced LMP1 protein levels. We also observed a decrease in intracellular level of this protein which correlated with a marked reduction of Bcl-2 expression in EBV-transformed B lymphocytes. We further demonstrated that anti-LMP1 sFv-mediated reduction of Bcl-2 correlated with increased sensitivity of these cells to drug-induced cell death. Therefore, these data suggest that an anti-LMP1 sFv used in combination with conventional chemotherapy may be useful for gene therapy of EBV-associated lymphomas in immunocompromised patients.

HIV-gp120 induced cell death in hematopoietic progenitor CD34+ cells.

Haematologic abnormalities accompany the majority of HIV-1 infections. At present it is unclear whether this is due directly to HIV infection of hematopoietic progenitor cells, or whether this results from an indirect mechanism secondary to HIV infection. Here we provide evidence for an indirect mechanism, whereby hematopoietic progenitor cells undergo HIV gp120-induced apoptosis (programmed cell death) even in the absence of HIV infection. Freshly isolated, purified human hematopoietic progenitor CD34+ cells, derived from both umbilical cord blood and bone marrow, co-expressed the CD4 marker at low density on their surface. Although these CD34+CD4+ cells theoretically should be capable of productive infection by HIV, we found that HIV-IIIB could not establish productive infection in these cells. Nonetheless, gp120 from IIIB could bind the cells. Thus, binding of gp120 did not correlate with infectivity. Furthermore, binding of gp120 was a specific event, leading to apoptosis upon crosslinking with anti-gp120 through a fas-dependent mechanism. If apoptosis is also observed in vivo even in uninfected hematopoietic cells, this could contribute significantly to the impairment in hematopoietic cell number and function. Our data suggest a novel indirect mechanism for depletion of CD34+ and CD34+-derived cells even in the absence of productive viral infection of these cells.

Gene therapy strategies for AIDS-related malignancies.

AIDS-related malignancies (ARM) include AIDS-defining cancers such as Kaposi's sarcoma, non-Hodgkin's lymphoma and cervical carcinoma. In addition, certain other malignancies are also increased with human immunodeficiency virus (HIV) infection. New antiretroviral agents and better prophylaxis and treatment of HIV-related opportunistic infections are prolonging the lives of HIV-infected individuals. There will thus likely be a continued rise in the incidence and prevalence of ARM in the long term, even if effective antiretroviral and other AIDS-related therapies reduce their appearance in the short term. There are presently no curative regimens for the common ARM, with the possible exception of some lymphomas. Survival is shortened by most, and treatment is often toxic and poorly tolerated. Gene therapies may thus offer a useful adjunct to conventional treatment strategies for selected ARM. Although some gene therapy strategies may work well in the HIV setting, the chronic viral infection, immunodeficient status of the host, the tendency for HIV-infected individuals to have altered drug metabolism and an increased rate of adverse drug reactions will likely present special challenges. This review summarizes the state-of-the-art in the fledgling field of gene therapy for ARM, and explores areas for future research.

CD4-mediated and CD8-mediated cytotoxic and proliferative immune responses to Toxoplasma gondii in seropositive humans.

Both CD4+ and CD8+ cytotoxic T lymphocytes (CTL) are part of the human immune response to Toxoplasma gondii infection. To further our understanding of Toxoplasma immunity, we investigated factors influencing stimulation of CD4+ or CD8+ human T. gondii-specific immune cells. Both antigen-pulsed and Toxoplasma-infected antigen-presenting cells (APC) induced cell proliferation. Toxoplasma-infected APC elicited strong proliferation of CD4+ cells, but little or no proliferation of CD8+ cells, unless high antigen loads were used. Toxoplasma-infected APC stimulated specific cytotoxicity poorly or not at all, owing to death of stimulated cultures, whereas antigen-pulsed APC strongly elicited specific cytotoxicity. Cytotoxicity elicited by either type of APC resided exclusively in CD4+ T cells in polyclonal cultures. Thus, Toxoplasma-infected APC elicited stronger CD4-mediated than CD8-mediated cell proliferation and generated CD4+ CTL more readily than CD8+ CTL. Nonetheless, specific CD8+ memory cells were demonstrated, and rare CD8+ Toxoplasma-specific CTL were subcloned. Fixed Toxoplasma-infected APC (which induce CD8+ CTL) also elicited cell proliferation, but polyclonal cultures stimulated with these infected APC did not die. Unfixed Toxoplasma-infected APC strongly inhibited phytohemagglutinin-induced cell proliferation, whereas fixed APC did not. These data suggested that infected APC were inhibitory or lethal to some immune cells. Further investigations into interactions between immune cells and Toxoplasma-infected cells likely will help elucidate factors involved in the immunopathogenesis of Toxoplasma infection. As other intracellular parasites, including Plasmodium spp. and Leishmania spp., also elicit CD4+ CTL, such work may help establish paradigms governing immunity to intracellular parasites.

HIV-specific cellular and humoral immune responses in primary HIV infection.

Primary human immunodeficiency virus (HIV) infection is characterized by a high-titer viremia that declines precipitously within weeks, most likely as a result of host immune responses. Peripheral blood mononuclear cells (PBMCs) and plasma of four recently HIV-infected individuals were examined to assess the humoral and cellular immune responses potentially involved in early suppression of viral replication. Neutralizing antibodies against autologous viral isolates were low or undetectable in three subjects studied. Cellular cytotoxicity was assayed using Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (B-LCLs) infected with recombinant vaccinia that express HIV-1 proteins. HIV envelope-specific cytotoxicity, which was not mediated by CD8+ cells nor human leukocyte antigen (HLA) class I restricted, developed in PBMCs of all four subjects early after primary infection, but was not correlated with declines in viremia. Gag-specific cytotoxic T lymphocyte (CTL) activity was observed in freshly isolated PBMCs of two subjects, and HIV-specific CTL cell lines were cultured from PBMCs of three subjects shortly after HIV infection. Antibody-dependent cellular cytotoxicity (ADCC) developed early in all four subjects, and was temporally correlated with declines in viremia in two subjects in whom viral load was well characterized. These data suggest that both CTL responses and ADCC may be critical to control of viral replication in acute HIV infection.

HIV-regulated diphtheria toxin A chain gene confers long-term protection against HIV type 1 infection in the human promonocytic cell line U937.

Gene therapy approaches have recently been investigated for the treatment of acquired immunodeficiency syndrome (AIDS), both in preclinical and clinical studies, because more traditional antiviral agents have proven to be of limited effectiveness. We have previously shown that long-term protection against both laboratory and clinical isolates of human immunodeficiency virus type 1 (HIV-1) was conferred by HIV-regulated diphtheria toxin A (DT-A) chain in a human T cell line. Because the monocyte/macrophage cell is an important reservoir for HIV-1 in infected individuals, we sought here to determine whether HIV-regulated DT-A would also be effective in the promonocytic cell line U937. We report here that long-term protection, conferred by HIV-regulated DT-A, was observed in U937 cells, but that protection was dependent on the stock of HIV IIIB used for challenge. HIV production was measured by p24 assays, polymerase chain reaction (PCR) for HIV vif, gag, and reverse transcriptase (RT) sequences, and cocultivation with peripheral blood mononuclear cells (PBMCs). Complete protection was seen in DT-A-transduced cells with a stock of IIIB propagated on H9 cells and titered on peripheral blood mononuclear cells (PBMCs), while protection in these same cells with a second stock of IIIB, propagated and titered on H9 cells, was only partial and dose dependent.

Optimization of gene transfer using cationic lipids in cell lines and primary human CD4+ and CD34+ hematopoietic cells.

Cationic lipids offer several advantages for gene delivery, both in vitro and in vivo. However, high-efficiency gene transfer has been demonstrated only for limited cell types. Here, we examine the level of expression of a luciferase reporter gene, delivered using cationic lipids, in both cell lines and primary human cells including peripheral blood mononuclear cells and CD34(+)-enriched hematopoietic cells. Variables shown to affect the efficiency of gene expression included the type of lipid, the amounts of DNA and lipid, the day of assay following transfection, the media used for lipid:DNA complex formation, the cell number, the promoter driving expression of the reporter gene and the physiological state of the cells (e.g., whether or not cells were differentiated). The maximal luciferase expression observed with the primary cells was one to two orders of magnitude lower than that seen in cell lines. Further studies, possibly involving altering the growth conditions for the cells, or using episomal vectors that will allow extrachromosomal maintenance of the DNA, are required to improve the level of transgene expression in the primary human cell types used here.

Cross-reactivity of human Toxoplasma-specific T cells: implications for development of a potential immunotherapeutic or vaccine.

Previous reports from this laboratory have demonstrated that human CD4+ Toxoplasma-specific cytotoxic T cell (CTL) clones generated by stimulation of peripheral blood mononuclear cells with Toxoplasma RH strain antigens also recognized target cells expressing C strain antigens. To extend these observations, additional Toxoplasma isolates were studied. A simple system for assessment of cytotoxicity using T cell lines rather than cloned CTL was used. Stimulation of human peripheral blood mononuclear cells with Toxoplasma RH strain antigens elicited cytotoxic T cell lines specific for target cells expressing antigens derived from many other Toxoplasma strains. Cell lines produced by stimulation with antigens derived from the related, nonpathogenic coccidian Besnoitia jellesoni were also cytotoxic for target cells expressing Toxoplasma antigens. Proliferative responses to many Toxoplasma isolates and to the Toxoplasma p30 protein were also noted.

Epstein-Barr virus-transformed B cells, a potentially convenient source of autologous antigen-presenting cells for the propagation of certain human cytotoxic T lymphocytes.

Antigen-specific cytotoxic T cells (CTL) are generally elicited in vitro by incubation of effector cells with an appropriate major histocompatibility complex-matched antigen-presenting cell (APC). In the case of CTL derived from inbred rodents, spleen cells from an animal of the same strain serve as a ready source of autologous major histocompatibility complex-identical APC. In outbred human populations, however, a convenient source of human leukocyte antigen-matched APC is ordinarily difficult to obtain, and for that reason human antigen-specific CTL may be difficult to propagate. We describe a method whereby Epstein-Barr virus-transformed human B cells (B-LCL) serve as a convenient source of efficient APC for the propagation of human antigen-specific CTL. B-LCL are produced by using B cells from the donor under study and are thus human leukocyte antigen identical to the donor. Using this method, we have propagated human CD4+ Toxoplasma gondii-specific CTL for up to 9 months in vitro, during which time the cells retained their functional capability.

Efficient foreign gene expression in Epstein-Barr virus-transformed human B-cells.

Epstein-Barr virus (EBV) is a herpesvirus that transforms B-cells (B-LCL) and has undergone intense scrutiny owing to its association with Burkitt's lymphoma, nasopharyngeal carcinoma, and immunoblastic lymphomas. B-LCL have also proven useful in the study of human immunology. We describe a novel system for inducing efficient foreign gene expression in B-LCL using biotinylated adenovirus as an endosome-disrupting agent. Plasmid DNA is coupled to the exterior of viral particles by streptavidin-polylysine chimeric proteins. Up to 67% of B-LCL may be induced to express foreign genes in vitro in transient expression systems, and gene expression lasts for at least 17 days. We have expressed firefly luciferase, beta-galactosidase (beta-gal), chloramphenicol acetyltransferase, HIV gag, and env genes, as well as infectious HIV, and the EBV-specific BZLF gene in B-LCL with this system. In vivo delivery of a beta-gal reporter gene to B-LCL was documented in a SCID mouse model. Potential applications include study of genetic regulation of EBV infection and transformation events, study of potential gene therapies for EBV-related B-cell tumors, and production of antigen-presenting cells for use in immunologic assays. Because of the high percentage of cells transformed and the length of foreign gene expression, the possibility of examining foreign gene expression in transient assays, without selection for clonal populations, exists.