Autoantibodies to MUC1 glycopeptides cannot be used as a screening assay for early detection of breast, ovarian, lung or pancreatic cancer.

BACKGROUND: Autoantibodies have been detected in sera before diagnosis of cancer leading to interest in their potential as screening/early detection biomarkers. As we have found autoantibodies to MUC1 glycopeptides to be elevated in early-stage breast cancer patients, in this study we analysed these autoantibodies in large population cohorts of sera taken before cancer diagnosis. METHODS: Serum samples from women who subsequently developed breast cancer, and aged-matched controls, were identified from UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) and Guernsey serum banks to formed discovery and validation sets. These were screened on a microarray platform of 60mer MUC1 glycopeptides and recombinant MUC1 containing 16 tandem repeats. Additional case-control sets comprised of women who subsequently developed ovarian, pancreatic and lung cancer were also screened on the arrays. RESULTS: In the discovery (273 cases, 273 controls) and the two validation sets (UKCTOCS 426 cases, 426 controls; Guernsey 303 cases and 606 controls), no differences were found in autoantibody reactivity to MUC1 tandem repeat peptide or glycoforms between cases and controls. Furthermore, no differences were observed between ovarian, pancreatic and lung cancer cases and controls. CONCLUSION: This robust, validated study shows autoantibodies to MUC1 peptide or glycopeptides cannot be used for breast, ovarian, lung or pancreatic cancer screening. This has significant implications for research on the use of MUC1 in cancer detection.

MVA-MUC1-IL2 vaccine immunotherapy (TG4010) improves PSA doubling time in patients with prostate cancer with biochemical failure.

PURPOSE: TG4010 is a recombinant MVA vector expressing the tumor-associated antigen MUC1 and IL2. We explored the effect two schedules of TG4010 on PSA in men with PSA progression. PATIENTS AND METHODS: A randomized phase II trial was conducted in 40 patients with PSA progression. Patients had PSA doubling times less than 10 months, with no overt evidence of disease. Patients received either weekly subcutaneous injection (sc) of TG4010 10(8) pfu for 6 weeks, then one injection every 3 weeks or sc injection of TG4010 10(8) pfu every 3 weeks. RESULTS: The primary endpoint of a 50% decrease in PSA values from baseline was not observed. Nevertheless, 13 of 40 patients had a more than two fold improvement in PSA doubling time. Ten patients had their PSA stabilized for over 8 months. Therapy was well tolerated. CONCLUSIONS: Although the primary endpoint was not achieved, there is evidence of biologic activity of TG4010 in patients with PSA progression, further investigation in prostate cancer is warranted.

A phase I trial of immunotherapy with intratumoral adenovirus-interferon-gamma (TG1041) in patients with malignant melanoma.

AIMS: Interferon-gamma (IFN-gamma) has been shown to upregulate MHC class I and II expression, and to promote generation of specific antitumor immune responses. We hypothesized that intratumoral administration of an IFN-gamma gene transfer vector facilitates its enhanced local production and may activate effector cells locally. We conducted a phase I dose-escalation study of a replication-deficient adenovirus-interferon-gamma construct (TG1041) to determine safety and tolerability of intratumoral administration, in advanced or locally recurrent melanoma. METHODS: Patients were enrolled at four successive dose levels: 10(7) infectious units (iu) (n=3), 10(8) iu (n=3), 10(9) iu (n=3), and 10(10) iu (n=2) per injection per week for 3 weeks. TG1041 was injected in the same tumor nodule weekly in each patient. Safety, toxicity, local and distant tumor responses and biologic correlates were evaluated. RESULTS: A total of 11 patients were enrolled and received the planned three injections per cycle. One patient with stable disease received a second cycle of treatment. A maximum tolerated dose was not reached in this study. No grade 4 toxicities were observed. Two grade 3 toxicities, fever and deep venous thrombosis were observed in one patient. The most frequently reported toxicities were grade 1 pain and redness at the injected site (n=8), and grade 1 fatigue (n=5) patients. Clinical changes observed at the local injected tumor site included erythema (n=5), a minor decrease in size of the injected lesion (n=5) and significant central necrosis by histopathology (n=1). Systemic effects included stable disease in one patient. Correlative studies did not reveal evidence of immunologic activity. CONCLUSION: Weekly intratumoral administration of TG1041 appears to be safe and well tolerated in patients with advanced melanoma.

Transduction of human dendritic cells with a recombinant modified vaccinia Ankara virus encoding MUC1 and IL-2.

The epithelial mucin MUC1 is considered an opportune target antigen for cancer immunotherapy, as it is over-expressed and exhibits aberrant glycosylation in malignant cells. Because dendritic cells (DC) are powerful initiators of immune responses, efforts have focused on tumor antigen-bearing DC as potent cancer vaccines. In this study we have characterized the transduction of monocyte-derived DC with a highly attenuated vaccinia virus vector [modified vaccinia Ankara (MVA)] encoding human MUC1 and the immunostimulatory cytokine IL-2. Analysis of transduced DC cultures generated from a number of donors revealed MUC1 expression in the range of 27-54% of the cells and a co-regulated secretion of bioactive IL-2. As shown by FACS analysis with MUCI-specific antibodies, the MVA-MUC1/IL-2-transduced DC predominantly expressed the fully processed glycoform of MUC1, typical of that displayed by normal epithelia. Over a 3-day period after transduction, transgene expression declined concurrent with an increase in MVA-induced cytopathic effects. The transduced DC stimulated allogeneic lymphocyte proliferation, indicating that DC immunostimulatory function is not impaired by vector transduction. In the presence of IL-2, MVA-transduced DC were able to enhance autologous lymphocyte proliferation. Also, vector expression was analyzed in DC cultures treated with TNF-alpha, a known DC maturation factor. As indicated by the up-regulation of several DC maturation markers, neither virus infection nor transgene expression influenced the maturation capacity of the cells. The MVA-MUC1/IL-2 vector effectively transduced both immature and TNF-alpha-matured DC. Overall, our results are encouraging for the clinical application of MVA-MUC1/IL-2-transduced DC.

Recombinant vaccinia viruses expressing immunoglobulin variable regions efficiently and selectively protect mice against tumoral B-cell growth.

The variable regions of the immunoglobulin (Ig) expressed on the surface of a malignant B cell can be considered tumor-specific antigens and, as such, could be targets for immunotherapeutic approaches. However, because until now the immunization procedures have been complex and have given only partial protection, it was necessary to find new methods of immunotherapy. Here, we present a successful method of vaccination against B-cell tumors in a murine model. We produced recombinant vaccinia viruses (rVV) expressing the heavy and the light chain of surface Ig of a patient's malignant B cells and we tested the ability of these rVV to protect immunized mice against tumor growth of transfectomas producing the same human Ig. The protection of the mice was complete and specific to the variable region of the immunizing heavy chain although specific lymphoproliferative and cytotoxic responses were not detectable in vitro. The protection was strictly dependent on the presence of CD4 T cells and asialo GM1+ cells. Furthermore, tumor protection clearly required gamma-interferon and was partially inhibited by blocking the Fas-Fas ligand interaction. We also show, in a murine syngeneic model, that rVV expressing a poorly mutated Ig protects against the growth of Ig-producing tumor.

Immunotherapy of established tumors in mice by intratumoral injection of an adenovirus vector harboring the human IL-2 cDNA: induction of CD8(+) T-cell immunity and NK activity.

Intratumoral (i.t.) injections of an adenovirus encoding the human interleukin-2 (IL-2) under the control of the RSV (Ad-pRSV-IL-2) or CMV (Ad-pCMV-IL-2) promoter were performed in established mastocytoma P815 tumors in B6D2 mice. Both early and long-term survival were found increased in mice treated with Ad-pCMV-IL-2 as compared with those obtained with Ad-pRSV-IL-2: tumor regression was observed in 30--50% of mice for the former and 5--15% for the latter. Difference in efficacy between the two vectors was directly correlated to the amount of IL-2 produced i.t. between 24 and 48 hours postinjection, which reached 10--20 ng/tumor for Ad-pCMV-IL-2 and 0.3--0.5 ng/tumor for Ad-pRSV-IL-2. In both cases, expression in the tumor was clearly detectable for a period of 7--10 days postinjection. Serum IL-2 was not detectable in mice treated with Ad-pRSV-IL-2, whereas expression peaked at a total of 1--2 ng at 24 hours but declined very rapidly in the Ad-pCMV-IL-2-treated group. Constant production of IL-2 inside the tumor was necessary for successful therapy because i.t. injections of recombinant IL-2 at levels up to 1 microg for five consecutive days did not lead to antitumoral activity. Evidence of induced systemic immunity following Ad-pCMV-IL-2 injections was obtained from rechallenge experiments in which tumor-free mice after treatment rejected a subsequent contralateral injection of a lethal dose of P815 tumor cells and from the observation that regression of nontreated tumors occurred in animals bearing bilateral tumors that were treated i.t. in a single tumor with Ad-pCMV-IL-2. P815-specific cytotoxic T lymphocytes (CTL) were found specifically in spleen cells from cured mice or rechallenged mice but not in control mice. Interestingly, limiting dilution analysis of anti-P815 CTL precursor (CTLp) frequency revealed a significant increase in mice cured of their tumor as compared to that obtained in naive mice or control mice treated or not with Ad-IL-2 but whose tumor was growing. In vivo depletion of T-cell subsets, as well as natural killer cells at the time of i.t. injections with Ad-pCMV-IL-2, demonstrated that both CD8(+) T cells and natural killer cells, but not CD4(+) T cells, were required for successful therapy. Finally, mice preimmunized with Ad-null viruses were severely compromised in their capacity to eradicate established P815 tumors after Ad-pCMV-IL-2 therapy, at least when neutralizing antibody titers reached a critical level.

Immune rejection of human dystrophin following intramuscular injections of naked DNA in mdx mice.

Intramuscular administration of plasmid expressing full-length human dystrophin in dystrophin-deficient adult mdx mice resulted in humoral and weak specific T cell responses against the human dystrophin protein. Following plasmid injection, human dystrophin was detected in the injected muscles at 7 days, but decreased thereafter. Anti-dystrophin antibodies were found 21 days following plasmid injection, which coincided with transient myositis. This immune rejection prevented the mice from expressing human dystrophin after a second plasmid injection. No anti-DNA antibodies were found. Anti-dystrophin antibodies were seen in a smaller proportion of plasmid-injected dystrophin-competent C57BL/10 mice, suggesting that the immune rejection of dystrophin may be explained partially by species differences in the dystrophin protein.

Recombinant vaccinia virus encoding human MUC1 and IL2 as immunotherapy in patients with breast cancer.

Polymorphic epithelial mucin, encoded by the MUC1 gene, is present at the apical surface of glandular epithelial cells. It is over-expressed and aberrantly glycosylated in most breast tumors, resulting in an antigenically distinct molecule and a potential target for immunotherapy. This transmembrane protein, when produced by tumor cells, is often cleaved into the circulation, where it is detectable as a tumor marker (CA 15.3) by various antibodies, allowing for early detection of recurrences and evaluation of treatment efficacy. The objective of the current study was to examine the clinical and environmental safety and immunogenicity of a live recombinant vaccinia virus expressing the human MUC1 and IL2 genes (VV TG5058), referred to here as TG1031. The study was an open-label phase 1 and 2 trial in nine patients with advanced inoperable breast cancer recurrences to the chest wall. The patients were vaccinated intramuscularly with a single dose of TG1031; three patients were treated at each of three progressive dose levels ranging from 5x10(5) to 5x10(7) plaque-forming units. A boost injection at their original dose level was administered in patients responding immunologically, clinically, or both. Vaccination resulted in no significant clinical adverse effects, and there was no environmental contamination by live TG1031. All patients had been vaccinated as children, and patients treated at the highest dose level mounted a significant anti-vaccinia antibody response. None of the nine patients had a significant increase in MUC1-specific antibody titers after one single injection, whereas five patients had a detectable increase in vaccinia virus antibody titers. Peripheral blood mononuclear cells of one patient at the intermediate dose level showed a proliferative response to in vitro culture with vaccinia virus, with a stimulation index of 6. A second patient treated at the intermediate dose level had a stimulation index of 7 to MUC1 peptide and of 14 after a boost injection. This patient had a concomitant decrease in carcinoembryonic antigen serum levels and remained clinically stable for 10 weeks. Evidence of MUC1-specific cytotoxic T lymphocytes was detected in two patients. Immunohistochemical analysis revealed an increase in T memory cells (CD45RO) in tumor biopsies after vaccination. The absence of serious adverse events, together with the documentation of immune stimulations in vivo, warrant the further use of TG1031 in immunotherapy trials of breast cancer.

Immunotherapy with mannan-MUC1 and IL-12 in MUC1 transgenic mice.

Mice immunised with oxidised mannan-MUC1 fusion protein (M-FP) develop MHC restricted CD8(+) cytotoxic T cells. We now demonstrate that in MUC1/HLA-A2 transgenic mice, IL-12 gives enhanced CTL, CTLp and tumor protection. CTLp in MUC1 transgenic mice with M-FP were 1/55,000, and with IL-12, this increased to 1/19,000, with improved tumor protection. Thus, IL-12 is important for effective CTL responses to MUC1 in transgenic mice.

The effect of T1 and T2 cytokines on the cytotoxic T cell response to mannan-MUC1.

MUC1 is a mucin over-expressed in breast cancer and a proposed target for immunotherapy. By immunising mice with MUC1 conjugated to mannan (M-FP), CD8(+) MHC-class-I restricted cytotoxic T lymphocytes (CTL), of high CTL precursor (CTLp) frequency (1/8000) and with significant tumour protection, can be induced. The effect of various cytokines [interleukin-2 (IL-2), IL-4, IL-6, IL-7, interferon gamma (IFNgamma), and granulocyte/macrophage-colony-stimulating factor (GM-CSF)] on the MUC1 CTL immune response was investigated (a) by measuring the frequencies of CTLp in mice immunised with vaccinia virus constructs containing recombinant cytokines and M-FP, or (b) by immunising cytokine- or cytokine-receptor-knockout (-/-) mice with M-FP. Vaccinia virus (VV) constructs containing recombinant cytokines were used either individually or in combination in vivo with M-FP immunisation. M-FP immunisations combined with VV-IL-2, VV-IL-7 and VV-GM-CSF, and combinations of VV-IFNgamma + VV-IL-2, VV-IFNgamma + VV-IL-4 or VV-GM-CSF + VV-IL-7 increased CTLp frequencies up to threefold (1/17 666: M-FP + VV-GM-CSF + VV-IL-7) compared to M-FP (1/77 500) alone. By contrast, M-FP combined with VV-IL-4 decreased the CTLp frequency threefold whereas VV-IL-6 and VV-IFNgamma had no effect. Studies in cytokine- and cytokine-receptor-gene-knockout (-/-) mice demonstrated that mice that are IL-2 -/- and IL-7 receptor -/- produce the same CTLp response to M-FP as do control mice, whereas responses in the IL-6 -/-, IL-10 -/- and IFNgamma -/- mice were marginally improved and responses to M-FP in IL-4 -/- and tumour necrosis factor receptor 2 -/- mice were weaker. In spite of the increase in CTLp frequency, this was not reflected in an in vivo tumour model. Tumour challenges using MUC1(+) P815 cells, demonstrated that the addition of cytokines had little additive effect on the already effective tumour-regression capabilities of M-FP alone.

MUC1-specific immune responses in human MUC1 transgenic mice immunized with various human MUC1 vaccines.

Analyses of MUC1-specific cytotoxic T cell precursor (CTLp) frequencies were performed in mice immunized with three different MUC1 vaccine immunotherapeutic agents. Mice were immunized with either a fusion protein comprising MUC1 and glutathione S-transferase (MUC1-GST), MUC1-GST fusion protein coupled to mannan (MFP) or with a recombinant vaccinia virus expressing both MUC1 and interleukin-2. Mouse strain variations in immune responsiveness have been observed with these vaccines. We have constructed mice transgenic for the human MUC1 gene to study MUC1-specific immune responses and the risk of auto-immunity following MUC1 immunization. Transgenic mice immunized with MUC1 were observed to be partially tolerant in that the MUC1-specific antibody response is lower than that observed in syngeneic but non-transgenic mice. However, a significant MUC1-specific CTLp response to all three vaccines was observed, indicating the ability to overcome T cell, but to a lesser extent B cell, tolerance to MUC1 in these mice. Histological analysis indicates no evidence of auto-immunity to the cells expressing the human MUC1 molecule. These results suggest that it is possible to generate an immune response to a cancer-related antigen without damage to normal tissues expressing the antigen.

Oxidised mannan antigen conjugates preferentially stimulate T1 type immune responses.

It is desirable to be able to produce either T1 or T2 responses and we have found that, in mice, mannose--coupled antigens stimulated T2 type responses antibodies and CTLs, whereas if oxidized, mannose--coupled antigens stimulated T1 responses little antibody and a potent CTL response. In addition, the cytokine profiles support the T1rT2 differentiation with these immunizations, in that oxidized mannan antigen gives IFNg, IL-2 and IL-12 production, whereas in the absence of oxidization, IL-4 and not the other cytokines is produced. A number of antigens have been examined--particularly Mucin 1 and the delivery method using mannose may be applicable to the other antigens.

Intratumoral injection of GM-CSF gene encoded recombinant vaccinia virus elicits potent antitumor response in a mixture melanoma model.

A recombinant vaccinia virus containing and expressing the gene for murine granulocyte-macrophage colony-stimulating factor (VVGM-CSF) was constructed and tested for its antitumor activity. A murine tumor model was established by injecting 10(5) B16F10 melanoma cells into the right rear leg of C57BL/6 mice. Three days after B16F10 inoculation, VVGM-CSF or a thymidine kinase gene-deficient vaccinia virus (VVTK) were injected intratumorly twice weekly for 3 weeks. The results showed that VVGM-CSF treatment significantly inhibited the growth of subcutaneous tumor and delayed the survival period of tumor-bearing mice. Splenic lymphokine-activated killer cell, natural killer cell, and cytotoxic T lymphocyte activities were not found to be altered after VVGM-CSF or VVTK therapy. Cytotoxic and phagocytic activity of peritoneal macrophages were found to be greatly elevated in mice treated with VVGM-CSF. Nitric oxide released from the macrophages was also increased. Considering these data, we may speculate that continuous secretion of GM-CSF and activation of macrophages may contribute to the antitumor effects of VVGM-CSF injected intratumorally.

Monoclonal anti-idiotypic antibody functionally mimics the human gastrointestinal carcinoma epitope GA733.

Anti-idiotypic antibodies (Ab2) that bind to the antigen-combining region of anti-tumor antibodies (Ab1) may functionally, and even structurally, mimic tumor antigen. We have previously demonstrated that polyclonal goat Ab2 directed against anti-human gastrointestinal carcinoma Ab1 GA733 induces anti-anti-idiotypic antibodies (Ab3) in animals that are Ab1-like in their binding specificity and idiotope expression. To obtain more defined Ab2 vaccines with potentially increased specificity and efficacy, a monoclonal Ab2 (FG1) was produced against Ab1 GA733 in rats. The monoclonal Ab2 FG1, similar to the polyclonal Ab2 described previously, induced Ab3 in rabbits that were Ab1-like in their idiotope expression and binding specificity to tumor cells and antigen. Antigen-specific Ab3 induced by Ab2 FG1 were easily detected in unprocessed rabbit sera, whereas the demonstration of such Ab3 after polyclonal Ab2 immunization required purification of the Ab3 from the rabbit sera. In addition, Ab2 FG1 induced antigen-specific humoral and cellular immunity in mice. Murine Ab3 bound specifically to antigen-positive tumor cells. Ab2-immunized mice showed antigen-specific delayed-type hypersensitivity (DTH) reaction, and cultured splenocytes from the immune mice demonstrated specific proliferation and cytokine (interferon-gamma and interleukin-4) secretion upon stimulation with GA733 antigen. However, immune mice were not protected against a challenge with syngeneic GA733 antigen-expressing colon carcinoma cells.

Active specific immunotherapy of pulmonary metastasis with vaccinia melanoma oncolysate prepared from granulocyte/macrophage-colony-stimulating-factor-gene-encoded vaccinia virus.

Vaccinia melanoma oncolysate (VMO) prepared with recombinant vaccinia virus encoding the gene of murine granulocyte/macrophage-colony-stimulating factor (GM-CSF) was tested for its therapeutic effect on melanoma pulmonary metastasis. The murine pulmonary metastasis model was established by injecting 2 x 10(5) B16F10 melanoma cells into the tail vein of a C57BL/6 mouse. Intraperitoneal injection of VMO was performed in tumor-bearing mice 3 and 10 days after B16F10 cell inoculation. The results showed that treatment with VMO prepared with GM-CSF-gene-encoded vaccinia virus (GM-CSFVMO) significantly decreased the number of murine pulmonary metastases and prolonged the survival of the tumor-bearing mice. Lymphocytes isolated from fresh blood and spleen of GM-CSFVMO-treated mice showed higher cytolytic activity against B16F10 melanoma cells when compared with lymphocytes from the mice of other treatment groups. Natural killer activity remained unchanged in the GM-CSFVMO-treated group. Cytotoxic activities of peritoneal macrophages were found to be greatly elevated in mice treated with GM-CSFVMO. Further study illustrated that the increased tumor necrosis factor and nitric oxide release from macrophages may contribute to their cytotoxic effects. These results suggest that the tumor oncolysate vaccine prepared with GM-CSF-gene-encoded vaccinia virus has a potent therapeutic effect on tumor metastasis through the efficient induction of antitumor immunity of the host, mainly through the cytotoxic effects of cytotoxic T lymphocytes and macrophages.

Directed cytokine expression in tumour cells in vivo using recombinant vaccinia virus.

Athymic (Swiss nude) and euthymic (DBA) tumour-bearing mice were injected intravenously with various vaccinia virus (Copenhagen strain) recombinants. Several days after inoculation, tumour cells were found to be well infected with infective vaccinia particles, while organs such as liver, spleen, brain and bone marrow showed barely detectable levels or no signs at all of virus infection. Injection of tumour bearing mice with recombinant VV harbouring the cDNA for either huIL-2 or muIL-6 resulted in detectable lymphokine in the sera of injected animals. Injection of tumour-bearing nude mice with VV-IL-6, but not with VV-IL-2, resulted in significant reduction in growth rate of the tumour, and in some cases, complete rejection of the tumour. Tumour-bearing euthymic mice responded differently. Intravenous injection of VV-IL-2, but not VV-IL-6 resulted in reduced growth rate of 50% of tumours and complete rejection of 17% of tumours.

Immunogenicity of HILDA/LIF either in a soluble or in a membrane anchored form expressed in vivo by recombinant vaccinia viruses.

Insertion of various cDNAs in the genome of the vaccinia virus (VV) enables the in vivo and in vitro study of the functional role and/or the immunogenicity of the virally encoded recombinant proteins. We have prepared a recombinant VV expressing the cDNA of the human cytokine HILDA/LIF (human interleukin for DA cells/leukaemia inhibitory factor), and used this virus to immunize mice against this protein, which is very homologous to its murine counterpart (approximately 80% homology). We also constructed and expressed by the same system a chimeric gene encoding the HILDA/LIF protein fused to the 37 COOH-terminal amino-acids of the human decay accelerating factor (DAF). This sequence proved to be sufficient for the targeting of the fusion protein to the cell membrane, where it is linked to the phosphatidylinositols. Both recombinant VVs induced cytokine-specific antibodies in mice as analysed with an ELISA where the recombinant HILDA/LIF was plastic-coated and a cytofluorometric assay where the LIF-DAF molecule was present at the cell surface of stably transfected P815. In the latter case HILDA/LIF remained biologically active suggesting that it was expressed in its native form. The LIF-DAF fusion protein was found to exhibit a better capacity to elicit an antibody response against the native form of the cytokine as detected in cytofluorometric assays. Whatever the recombinant virus used to immunize the mice, the MoAbs obtained were positive either in the ELISA or in the cytofluorometric assays but one, which suggested that the plastic coating induced a conformational change of HILDA/LIF.

In vivo delivery of interleukin-6 using vaccinia virus: effects on T lymphocytes in nude mice.

We have constructed a recombinant vaccinia virus (VV) expressing the human interleukin-6 (IL-6) gene, VV(IL-6). After injection of VV(IL-6) i.v. into Balb/c mice, circulating IL-6 was detected during 3 days with the peak activity on day 4, indicating that VV injection is an effective method to deliver lymphokines in vivo. We have further examined the effects of IL-6 in vivo in immunodeficient mice. Nude mice were injected i.v. with VV(IL-6). Ten days after the injection, mice were sacrificed and spleen cells were obtained. Spleen cells from VV(IL-6) injected mice proliferated remarkably in response to IL-2, while spleen cells from mice injected with unrelated VV manifested no particular proliferation in response to lymphokines. When spleen cells were further cultured in vitro for 5 days in the presence of Concanavalin-A stimulated rat spleen cell supernatant (Con-A factor), CD4 or CD8 positive cells were detected in the VV (IL-6) injected group, while few positive cells were detected in the control groups. These results suggest that IL-6 stimulates nude mice spleen cells in vivo, to a stage where they are able to proliferate in response to IL-2, or to differentiate into CD4 or CD8 positive cells in presence of rat Con-A factor.

DNA binding factors that bind to the negative regulatory element of the human immunodeficiency virus-1: regulation by nef.

The nef gene has been reported to be a silencer of human immunodeficiency virus (HIV) transcription that requires the presence of the negative regulatory element (NRE) located at the 5' end of the HIV long terminal repeat (LTR) to exert its negative effect. We have examined nuclear extracts from human, nontransformed T cells for factors that bind to the NRE of HIV-1 and to determine whether binding of factors to this region can be affected by the nef gene. Using gel retardation and methylation interference assays, we have observed several DNA binding factors that bind to a region between nucleotides -315 and -240 upstream of the cap site, within the NRE segment of the 5' LTR. Furthermore, the precise locations of the binding sites for two of these factors, termed here A1 and R, were determined. Factor A1 appears to belong to a family of cellular activation associated factors (called here A1-A4), but it is distinct in that it is the only DNA binding factor so far observed that appears to be downregulated by the nef gene or its product and that it has been found only in cells undergoing lymphokine-driven cell division. In contrast to the A factors, factor R appears to be associated with cellular quiescence and binds to a nearby but distinct site in the NRE. Experiments in which extracts were mixed before gel retardation suggest that the binding of factors R and A1 are mutually exclusive. Based on these observations we propose a model in which the nef gene aids in the maintenance of HIV latency by downregulating the binding of proliferation associated DNA binding factor, which we have called A1.

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.