Gene therapy approaches for the management of non-small cell lung cancer.

Targeting the specific genetic lesions responsible for carcinogenesis and cancer progression is an attractive strategy for developing more effective anticancer therapies and reducing treatment-related toxicity. The restoration of defective tumor suppressor gene pathways by replacement of tumor suppressor genes in cancer cells has been studied in lung cancer. The most extensively studied agent is the wild-type p53 tumor suppressor gene delivered by an adenoviral vector. Clinical trials to date in non-small cell lung cancer and head and neck cancer have consistently shown evidence of gene transduction and expression, mediation of apoptosis, and clinical responses including pathologic complete responses. However, it also is clear that this approach can be improved. Promising avenues for investigation include improved gene delivery systems, induction of bystander effects, and adjuvant use of gene therapy with conventional chemotherapy, radiation therapy, and surgery. However, these strategies will need further refinement to succeed clinically. This review examines several important issues in cancer gene therapy in general and the most recent achievements in gene therapy for non-small cell lung cancer.

P53 gene replacement for cancer--interactions with DNA damaging agents.

Clinical trials of p53 gene replacement have provided information that will be useful in the design of future gene therapy strategies. Direct intratumor injection has low toxicity and thus can be readily combined with existing treatments. Post-injection gene expression can be documented and occurs in the presence of an anti-adenovirus immune response. Importantly, this treatment can cause tumor regression or prolonged stabilization. Future research directions will include development of more efficient vectors, use of novel genes, and combined modality approaches. Unresectable tumors are a prominent problem in oncology, with proven therapies such as radiotherapy and chemotherapy controlling less than 20% of lung cancers. Based on the preclinical and clinical studies discussed, it now seems that these conventional therapies may provide renewed potential when used in conjunction with transfer of a functional p53 gene.

Gene replacement strategies for treating non-small cell lung cancer.

Fewer than 15% of the 170,000 patients who develop lung cancer each year will survive their disease, which shows the need for novel, more specific, and less toxic therapeutic strategies. Recent advances in molecular biology have made it possible to ascertain which genetic alterations contribute to the etiology of cancer. For example, the tumor-suppressor gene, p53, responsible for directing repair of damaged DNA or committing a cell to apoptosis, is mutated or otherwise altered in more than 50% of cancers, including 40% to 70% of non-small cell lung cancers. Many p53-deficient tumors have proven remarkably resistant to radiotherapy and chemotherapy. The preclinical and clinical studies of gene therapy reviewed in this article show (1) successful transfer and expression of a potentially therapeutic p53 gene construct in tumor cells, (2) observation of antitumor effects in vitro and in vivo, and (3) most critically, a lack of significant toxicity. The results of these studies indicate that gene replacement therapy is a feasible alternative therapy for cancer. In addition, these studies show that transfer of the p53 gene can induce radiation sensitization in previously radiation-resistant tumors, leading to the possibility of new therapeutic protocols combining gene replacement with radiation therapy.

Role of F4/80+ cells during induction of hapten-specific contact hypersensitivity.

F4/80, a monoclonal antibody that binds to a surface molecule on mature macrophages and certain dendritic cells, has been used to explore the role of epidermal and dermal cells as antigen-presenting cells (APC) during the induction of contact hypersensitivity (CH) in mice. Systemic administration of the antibody appeared to have little or no physical or functional effect on intraepidermal Langerhans' cells, even though a subpopulation of these cells expressed the F4/80 ligand. None the less, systematically administered F4/80 antibodies were able to impair CH induction when dinitrofluorobenzene (DNFB) was painted on normal body wall skin of BALB/c mice [an ultraviolet B (UVB)-resistant strain]. Interestingly, systemic F4/80 antibodies did not affect CH induction in C57BL/10 mice (a UVB-susceptible strain). When a sensitizing dose of hapten was injected intracutaneously (i.c.) into F4/80-treated BALB/c and C57BL/10 mice, CH induction was impaired in both inbred strains, although the severity of impairment was greater in BALB/c mice. Following UVB radiation of body wall skin, anti-F4/80-treated BALB/c mice displayed very feeble CH, whether hapten was painted epicutaneously or injected i.c. at the irradiated site. Based on these and other recent reported results, it is concluded that (1) BALB/c mice rely partially upon dermal, F4/80+ cells as a source of APC when hapten is applied epicutaneously, whereas C57BL/10 mice rely almost exclusively upon epidermal Langerhans' cells in this circumstance; and (2) after UVB radiation of skin, BALB/c mice can use F4/80+ dermal cells as the source of APC function when hapten is painted epicutaneously. These findings are discussed with respect to the cellular basis for the differential susceptibilities of genetically defined strains of mice to the deleterious effects of UVB radiation on CH induction.

Fresh and cultured Langerhans cells display differential capacities to activate hapten-specific T cells.

Langerhans cells (LC) that have been cultured for 3 days acquire potent T cell-activating properties when compared to freshly prepared, uncultured LC. By contrast, fresh LC are superior to cultured LC in the ability to process native protein Ag. To define further the disparate functional properties of these epidermally derived APC, freshly isolated and cultured epidermal cells (EC) enriched for LC were prepared from BALB/c mice. Highly purified T cells from naive mice, and from mice sensitized epicutaneously with dinitrofluorobenzene, have been examined for their capacity to respond to fresh and cultured EC; 1) in the presence of staphylococcal enterotoxin B; and 2) after the EC had been derivatized with dinitrofluorobenzene. Both fresh and cultured EC activated syngeneic T cells in the presence of staphylococcal enterotoxin B, and fresh and cultured DNP-derivatized EC induced proliferation among DNP-specific T cells. Only cultured, hapten-derivatized EC were able to activate unprimed syngeneic T cells in vitro, and these cells responded as though "primed" when re-exposed to DNP-derivatized spleen cells in secondary cultures. In addition, naive lymphocytes that were activated by cultured DNP-EC were able to evoke local contact hypersensitivity reactions when injected into the pinnae of naive mice that were then painted with dinitrofluorobenzene. By contrast, naive syngeneic T cells exposed to fresh DNP-EC neither proliferated nor differentiated into effector cells. We conclude that fresh LC can constitutively activate primed, but not unprimed, hapten-specific T cells, whereas cultured LC readily both primed and unprimed T cells. The capacity of hapten-derivatized cultured EC to convert naive, hapten-specific T cells into cells that mediate contact hypersensitivity supports the proposal that cultured LC are the functional equivalents of epidermal LC that have migrated to draining lymph nodes. The ability of hapten-derivatized fresh LC to activate primed, hapten-specific T cells is consistent with the view that fresh LC are functionally equivalent to LC within the epidermis.

Effects of chloroquine on antigen-presenting functions of epidermal cells from normal and psoriatic skin.

The lysosomotropic drug chloroquine has been added to cultures containing peripheral blood mononuclear cells (PBMC) and allogeneic antigen-presenting cells obtained from the epidermis of normal human skin or from skin of patients with psoriasis. We found that in the presence of chloroquine, the allostimulatory properties of freshly obtained, normal epidermal antigen-presenting cells (EAPC) were profoundly impaired. By contrast, normal EAPC (cultured for 72 h prior to exposure to alloreactive T cells), as well as fresh EAPC from psoriatic skin were not impaired by chloroquine. In fact, in some experiments, cultured EAPC and psoriatic EAPC in the presence of chloroquine displayed significantly enhanced abilities to activate allogeneic T cells. Moreover, chloroquine partially reversed the inhibitory effect of transforming growth factor-beta (TGF beta) on T-cell activation induced by cultured normal EAPC. Fresh normal EAPC, which are normally impervious to the effects of TGF beta, were not protected by TGF beta from chloroquine inhibition. We conclude that the addition of chloroquine to tissue culture medium unmasks important differences in antigen processing and presenting properties of fresh, normal EAPC, on the one hand, and cultured normal EAPC and psoriatic EAPC on the other. The ability of chloroquine to exaggerate the accessory cell function of the latter cells may relate to the capacity of this drug to cause the secretion of acid hydrolases into their immediate microenvironment. Moreover, the capacity of chloroquine to enhance the accessory cell functions of freshly obtained psoriatic EAPC emphasizes an abnormality that psoriatic cells in the epidermis constitutively express. We postulate that this abnormality may be related to the clinical observations that psoriatic skin lesions may be induced or aggravated by chloroquine therapy.

T-lymphocyte-activating properties of epidermal antigen-presenting cells from normal and psoriatic skin: evidence that psoriatic epidermal antigen-presenting cells resemble cultured normal Langerhans cells.

Fresh and cultured human Langerhans cells display disparate functional programs, based on their capacities to activate autologous and allogeneic T cells, and with respect to their susceptibility to inhibition by transforming growth factor-beta (TGF beta). We have compared the functional properties of epidermal antigen-presenting cells (APC) procured from uninvolved and involved skin of patients with psoriasis with fresh and cultured normal epidermal cells. Freshly obtained psoriatic epidermal APC resembled cultured normal epidermal cells in their superior capacity to activate syngeneic and allogeneic T cells; fresh normal epidermal cells failed to activate syngeneic T cells, and induced only modest proliferation among allogeneic T cells. The modest T-cell--activating properties of fresh, normal epidermal cells were not suppressed by TGF beta, whereas the T-cell--activating potential of psoriatic epidermal cells, cultured normal epidermal cells, and blood APC was inhibited approximately 50% by TGF beta. Thus, fresh psoriatic epidermal APC resemble cultured normal epidermal cells functionally. Because these properties are already evident in cells obtained from uninvolved psoriatic skin, the "cultured" functional phenotype of epidermal APC in this disease may precede the appearance of active psoriatic skin lesions. Surface marker analysis of normal and psoriatic epidermal cell suspensions revealed that virtually all of the bone marrow--derived cells in normal epidermal cell suspensions were conventional (CD1+) Langerhans cells, whereas CD1+ cells comprised only a minority of bone marrow--derived (CD45+) cells in psoriatic epidermis. It is speculated that some of the CD1-, CD45+ cells in psoriatic epidermis may be Langerhans cells that have lost their "fresh" phenotype. These data indicate that an abnormality in epidermal APC function exists in psoriatic skin--even before clinical lesions develop, and we speculate that the abnormal capacity of psoriatic epidermal APC to activate syngeneic T cells may be important in the expression of keratinocyte pathology. Because psoriatic epidermal APC functions were profoundly inhibited in vitro by treatment with cyclosporin A, the effectiveness of this drug in psoriasis may be due in part to its ability to inhibit epidermal antigen-presenting cell function in vivo.

Comparison of effects of transforming growth factor-beta and cyclosporin A on antigen-presenting cells of blood and epidermis.

The antigen-processing and -presenting functions of freshly obtained epidermal Langerhans cells (fresh LC) and 72-h cultured Langerhans cells (cultured LC) differ remarkably. It has been proposed that the disparate functional programs revealed in vitro may correspond directly with distinct in vivo physiologic functions--fresh LC are the in vitro equivalent of intraepidermal LC and cultured LC are equivalent to LC that have migrated from skin to the draining lymph node. As an approach to studying this proposal, we have compared the effects of two immunosuppressive agents, cyclosporin A (CsA) and transforming growth factor-beta (TGF beta), on the alloantigen-presenting capabilities of fresh LC, cultured LC, and peripheral blood mononuclear cells (PBMC). CsA pretreatment (1 and 10 mu/ml x 2 h) profoundly inhibited alloantigen presentation by fresh LC, cultured LC, and PBMC. By contrast, TGF beta pretreatment (1 and 10 ng/ml x 2 h) inhibited presentation by PBMC and cultured LC, but not by fresh LC. The resistance of fresh LC to the deleterious effects of TGF beta is discussed in terms of the possibility that TGF beta may inhibit antigen processing following conventional endocytosis. We suggest that fresh, but not cultured, LC escape TGF beta effects because they possess an "alternative" endocytic pathway, marked by the presence of Birbeck granules.

Functional dichotomy between Langerhans cells that present antigen to naive and to memory/effector T lymphocytes.

The general thrust of this volume is to review the roles of accessory cells in regulating T and B lymphocytes. To that end, we have summarized the evidence that indicates the crucial role that Langerhans cells play in the induction and expression of immunity to antigens that gain access to, or arise within, skin. Langerhans cells accomplish this important goal by their abilities to (a) activate naive T cells to antigens not previously encountered by the host, and (b) activate memory/effector T cells specific for previously encountered antigens. Arguments have been advanced to support the view that the functional properties of Langerhans cells used to present antigens to naive T cells differ substantially from the properties that equip Langerhans cells to activate effector T cells. The arguments are based in part on the fact that Langerhans cells carry out these functions in two very different environments: in the epidermis, and in the draining lymph node. The arguments are also based on results of in vitro experiments that reveal distinct differences in antigen processing and presenting properties of Langerhans cells freshly obtained from mouse and human skin as compared to Langerhans cells that have been cultured in vitro for 2-3 days. We propose that freshly explanted Langerhans cells faithfully reflect the functional program of intraepidermal Langerhans cells, and are able to present antigen to memory/effector T cells that enter the epidermal compartment. To accomplish this task, epidermal LC pick up environmental antigens, process them with great efficiency, and then present them in situ, without further upregulation of "accessory" signals (cell-adhesion molecules, secretion of additional cytokines). They can carry out this function, even in the presence of TGFB--a a cytokine which is constitutively made by keratinocytes, and which we have found to profoundly inhibit antigen presentation by most other types of "professional" antigen-presenting cells. Intraepidermal Langerhans cells are also capable of carrying cutaneous antigens through the dermal epidermal junction and migrating to the draining lymph node. We further propose that cultured Langerhans cells are fated to present antigens to unprimed/naive T cells, and thereby to initiate immune responses to new cutaneous antigens. Cultured LC process antigens less efficiently than fresh cells, but their unique capacity to present antigen effectively to unprimed T cells rests chiefly on the fact that they have significantly upregulated cell surface adhesion molecules, expression of MHC molecules, and secretion of activating cytokines--the "accessory" signals that are required for arousing naive T cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of an HSV-1/HSV-2 cross-reactive T cell determinant.

The results from a number of studies have documented that the HSV glycoprotein gD is an important target for neutralizing antibodies. In contrast, little is known about the Th cell determinants present on HSV that are required for anti HSV gD antibody production. In our study we have immunized BALB/c mice with a recombinant source of HSV-1 gD lacking the carboxyl-terminal 93 amino acids. T cell hybridomas produced from the immunized animals recognized a single antigenic peptide (amino acids 246-261) in the context of I-Ad. The determinant expressed by gD peptide 246-261 was generated and presented by both HSV-1 and HSV-2 infected APC. Fine specificity analysis using truncated synthetic gD peptides revealed that the minimal amino acids recognized by the T hybrids were identical between HSV-1 and HSV-2. In addition, the minimal peptide-I-Ad binding analysis demonstrated that the minimal peptide sequence required for the binding to I-Ad and for T cell recognition contained two prolines. Thus, this important HSV antigenic determinant would not be expected to form an amphipathic alpha-helix and could therefore be missed by algorithms currently used to predict which amino acid sequences would be antigenic based on the propensity to form helices.

Influence of microenvironmental factors on human Langerhans cell function in vitro.

When murine epidermal cells are placed in tissue culture for 48-72 hr, the Langerhans cell subpopulation acquires a markedly enhanced capacity to activate autologous as well as allogeneic T cells. This change is mediated largely, if not exclusively, by GM-CSF derived from keratinocytes present in the same cultures. When human epidermal cells are cultured under similar conditions, Langerhans cells display much more limited functional changes. We have investigated whether the relative failure of human Langerhans cells to change their functional program in vitro is due to (a) production of prostaglandins by keratinocytes during functional assays (which would inhibit T cell proliferation), and/or (b) relative deficiency of GM-CSF in the 72-hr epidermal cell culture. Freshly procured and cultured (72 hr) human epidermal cells were used as stimulators for allogeneic T cells in cultures to which indomethacin was added to block prostaglandin production. Under these circumstances, the allostimulatory capacity of cultured cells was enhanced, although there was no significant increase in the stimulatory properties of fresh cells, implying that prostaglandins may account in part for the relatively poor antigen presenting properties of cultured human cells. In addition, human epidermal cells were cultured with or without exogenous GM-CSF prior to being used as stimulators for autologous and allogeneic T cells. Following exposure to GM-CSF, cultured human epidermal cells acquired markedly enhanced T cell-activating properties, rivaling those reported for cultured murine epidermal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro evidence that Langerhans cells can adopt two functionally distinct forms capable of antigen presentation to T lymphocytes.

Monodisperse suspensions of epidermal Langerhans cells (LC) have been examined for their capacity to process and present Ag immediately upon extraction from mouse epidermis (fresh LC) and after 72 h in tissue culture (cultured LC). Cultured, but not fresh, LC stimulated proliferation among autologous T cells, whereas fresh, but not cultured, LC proved to be superior at processing native OVA for presentation to an OVA peptide-specific, MHC-restricted T cell hybridoma. Cultured LC were also more effective at stimulating proliferation among allogeneic T cells. However, a significant, but difficult to quantify, component of lymphocyte activation in these assays was derived from the ability of cultured LC to stimulate autologous T cells. It has been proposed that the superior capacity of cultured LC to stimulate T cells in these assays is due to the "immaturity" of freshly prepared LC--which "mature" during the 72-h culture interval. Based on the observation that fresh LC are superior at processing native protein Ag, we would amend the currently held notion that there is a "precursor-product" relationship between fresh and cultured LC to include the fact that these populations are differentially equipped to carry out distinct physiologic functions and that fresh LC should not, therefore, be considered "immature." We propose that fresh LC (in vitro equivalents of intraepidermal LC) can process native protein Ag with great efficiency, and can present these Ag in situ to memory and effector T cells (high affinity TCR interactions). Cultured LC (in vitro equivalents of LC that have migrated from skin to draining lymph node) exchange highly efficient Ag processing for acquisition of accessory molecules (surface ligands and secreted cytokines) that promote activation of unprimed T cells (including even low affinity TCR interactions).

Studies on the capacity of B cells as well as T cells to serve as accessory cells for the activation of herpes simplex virus-specific cytolytic T cells.

Experiments were conducted in an effort to determine the ability of B and T lymphocytes to serve as APC for the activation of HSV-primed splenic T cells to become class I-restricted, HSV-specific CTL. The results showed that both freshly isolated splenic B cells as well as LPS and dextran sulfate (L/D)-activated B cells were effective at stimulating the generation of CTL during a 5-day in vitro culture. There was no requirement for the addition of exogenous IL-2 to the culture and, since murine B cells do not appear to express either membrane or secreted IL-1, this lymphokine appears to either not be required for the activation of virus-specific CTL or to be provided by the T cells themselves. When normal B cells were separated into fractions enriched for resting vs activated cells and then tested for their ability to stimulate the generation of HSV-specific CTL, it was found that while the activated B cells were quite effective at stimulating the generation of CTL, resting B cells were ineffective at carrying out this function. In contrast to normal B cells, normal T cells were unable to act as APC. However, Con A-activated T lymphoblasts were equivalent to L/D B cells in their ability to mediate the generation of CTL activity. L/D B cells that had been pulsed with HSV and then incubated at 37 degrees C for greater than 1 h could be fixed with paraformaldehyde and were still able to function as APC. The finding that L/D B cells, that had been fixed at 1 h or less after exposure to HSV, were unable to function as APC suggested that either active Ag "processing" steps may be required for the presentation of Ag in the context of class I molecules or that there is a requirement for the synthesis of viral protein Ag before presentation.

Ia-specific mixed leukocyte reactive T cell hybridomas: analysis of their specificity by using purified class II MHC molecules in synthetic membrane system.

This study was undertaken to determine the nature of the antigens recognized in allogeneic and syngeneic mixed leukocyte reactions (MLR). Specifically, we wished to determine whether Ia antigens alone were recognized by MLR-reactive T cells, or whether the specificity was determined by the corecognition of non-MHC antigens together with syngeneic or allogeneic Ia. To do this we used 11 T cell hybrids that were characterized as being specific for Iad and were tested their capacity to respond to isolated I-Ad or I-Ed that had been incorporated into liposomes and had bound to the surface of glass beads. Of nine alloreactive T cell hybrids (five I-Ad-and four I-Ed-specific), seven were shown to be responsive to the relevant isolated Ia antigen on glass beads. Also, two of two syngeneic I-Ad-specific T cell hybrids responded to I-Ad on the glass beads. One of the two alloreactive T cell hybrids that failed to respond to the relevant Ia antigen on glass beads was shown to be specific for an antigen in fetal calf serum (FCS) that was recognized in the context of the allo-Ia antigen (I-Ed), because when intact accessory cells were used, a response by this hybrid was only observed when FCS was present in the assay culture medium or when the accessory cells were pre-pulsed with FCS. The possible involvement of FCS antigens and non-Ia accessory cell antigens in the stimulation of the nine T cell hybrids that responded to isolated Ia on glass beads was evaluated. T cell hybrids that were grown and were tested in serum free medium were still capable of reacting to Ia on beads. The isolated Ia preparations used were greater than 90% pure, and their capacity to stimulate the T cell hybrids did not correlate with the degree of contamination with non-Ia proteins. We conclude from these studies that the majority of T cells that respond to allogeneic or syngeneic Ia bearing stimulator cells are specific for the Ia antigens themselves, and do not require the co-recognition of other non-Ia antigens; nor is there any requirement for Ia antigen processing for this recognition.

Antigen presentation by splenic B cells: resting B cells are ineffective, whereas activated B cells are effective accessory cells for T cell responses.

In this study, we have investigated the ability of splenic B cells to act as antigen-presenting cells. Previous data had established that lipopolysaccharide (LPS)-activated B cells were effective antigen-presenting cells; however, the relative capacity of resting B cells to carry out this function remains controversial. Splenic B cells from naive BALB/c mice were depleted of macrophages, dendritic cells, and T cells, and were fractionated on the basis of cell density by using Percoll gradient centrifugation. Fractions were collected from the 50/60, 60/65, and 65/72% interfaces and from greater than 72% (pellet). Cytofluorograph analysis of the fractionated B cells showed that the two lower density fractions (50/60 and 60/65) contained a number of cells which, by cell size determination, appeared to be activated B cells, whereas the two higher density fractions (65/72 and greater than 72) appeared to contain predominantly small resting B cells contaminated by many fewer activated B cells. Functionally, the capacity of fractionated B cells to act as accessory cells for a concanavalin A response or present the antigens chicken ovalbumin (OVA) or OVA-tryptic digest gave similar results, which indicated a striking hierarchy of accessory cell function in the different Percoll fractions. When normalized to the most active low-density fraction (50/60%), the activity of the other fractions were: 60/65 = 78%; 65/72 = 25%; and greater than 72 = 4%. The differences in the functional capacity between the various Percoll fractions did not appear to be due to differences in Ia expression. Although the expression of Ia varied approximately 12-fold within any one fraction, there was little difference in the mean amount of Ia on cells obtained from the various fractions. Kinetic studies showed that activation of B cells with LPS and dextran sulfate resulted in the expression of two stages of functional development. The first stage was an increased efficiency of accessory cell function that was abrogated by irradiation with 4000 rad followed by a second stage, which was characterized by the acquisition of resistance to treatment with 4000 rad. When nonfractionated B cells that had been stimulated with LPS and DexSO4 were sorted on the basis of cell size into a small B cell fraction and a large B cell fraction, only the large B cells were able to present antigen. Taken together, these data suggest that much of the accessory cell function associated with splenic B cells can be accounted for by the relatively small percentage of activated B cells present in the spleen.(ABSTRACT TRUNCATED AT 400 WORDS)