Preclinical evaluation of "whole" cell vaccines for prophylaxis and therapy using a disabled infectious single cycle-herpes simplex virus vector to transduce cytokine genes.

The development of genetically modified "whole" tumor cell vaccines for cancer therapy relies on the efficient transduction and expression of genes by vectors. In the present study, we have used a disabled infectious single cycle-herpes simplex virus 2 (DISC-HSV-2) vector constructed to express cytokine or marker genes upon infection. DISC-HSV-2 is able to infect a wide range of tumor cells and efficiently express the beta-galactosidase reporter gene, granulocyte-macrophage colony-stimulating factor (GM-CSF), or IL-2 genes. Gene expression occurred rapidly after infection of tumor cells, and the level of production of the gene product (beta-galactosidase, GM-CSF, or IL-2) was shown to be both time-and dose-dependent. Vaccination with irradiated DISC-mGM-CSF or DISC-hIL-2-infected murine tumor cells resulted in greatly enhanced immunity to tumor challenge with live parental tumor cells compared with control vaccines. When used therapeutically to treat existing tumors, vaccination with irradiated DISC-mGM-CSF-infected tumor cells significantly reduced the incidence and growth rates of tumors when administered locally adjacent to the tumor site, providing up to 90% protection. The prophylactic and therapeutic efficacy of DISC-mGM-CSF-infected cells was shown initially using a murine renal cell carcinoma model (RENCA), and the results were confirmed in two additional murine tumor models: the M3 melanoma and 302R sarcoma. Therapy with DISC-infected RENCA "whole" cell vaccines failed to reduce the incidence or growth of tumor in congenitally T-cell deficient (Nu+/Nu+) mice or mice depleted of CD4+ and/or CD8+ T-lymphocytes, confirming that both T-helper and T-cytotoxic effector arms of the immune response are required to promote tumor rejection. These preclinical results suggest that this "novel" DISC-HSV vector may prove to be efficacious in developing genetically modified whole-cell vaccines for clinical use.

Regulation of T cell activation in vitro and in vivo by targeting the OX40-OX40 ligand interaction: amelioration of ongoing inflammatory bowel disease with an OX40-IgG fusion protein, but not with an OX40 ligand-IgG fusion protein.

OX40 is a member of the TNFR superfamily, and is found predominantly on activated CD4-positive T cells. In vitro an OX40-IgG fusion protein inhibits mitogen- and Ag-driven proliferation and cytokine release by splenocytes and lymph node T cells. In contrast, an OX40 ligand-IgG fusion protein enhanced proliferative responses. In normal mice, OX40-positive cells are observed only in lymphoid tissues, including Peyer's patches of the gut. In mice with hapten-induced colitis or IL-2 knockout mice with spontaneous colitis, OX40-positive cells are found infiltrating the lamina propria. Administration of the OX40-IgG fusion protein to mice with ongoing colitis (but not the OX40 ligand-IgG) ameliorated disease in both mouse models of inflammatory bowel disease. This was evidenced by a reduction in tissue myeloperoxidase; reduced transcripts for TNF-alpha, IL-1, IL-12, and IFN-gamma; and a reduction in the T cell infiltrate. Targeting OX40 therefore shows considerable promise as a new strategy to inhibit ongoing T cell reactions in the gut.

A multiparameter flow cytometric method to study surface molecules involved in interactions between subpopulations of cells.

The interactions between T and B lymphocytes are mediated by several antigen-independent adhesion molecules including LFA-1/ICAM-1 and CD2/LFA-3. Recently new pairs of adhesion molecules involved in T and B interactions have been described: CD28/B7, CD5/CD72 and CD45RO/CD22. In order to study these heterotypic adhesion events, the phenotypes of the subpopulations as well as new potential adhesion molecules involved in conjugate formation, we have developed a flow cytometric method which analyses conjugate formation between T and B cells. The two types of cells were loaded with two vital intracellular dyes: human T lymphocytes purified from blood or tonsils were labelled with BCECF-AM (green fluorescence) and the B lymphoblastoid cell line, RPMI 8866 was labelled with Indo-1-AM (blue fluorescence). The two labelled cell populations were mixed, gently centrifuged for 5 min and then incubated at 37 degrees C in a waterbath for 5 min. The cells were then gently resuspended by inversion and analysed with a double laser flow cytometer. This method permitted us to discover new molecular interactions since preincubation of the two populations with monoclonal antibodies directed against some surface molecules inhibited conjugate formation. As an example, using this technique we found that the low affinity IgE receptor, CD23 and the CR2/EBV receptor are involved in T cell/B cell adhesion and can therefore be considered as a new pair of adhesion molecules. This method also seems to be applicable to recombinant cells bearing a single adhesion molecule such as LFA-1 and ICAM-1. A particular advantage of the two intracellular dyes we used is that they are compatible with the dyes commonly used for classical simultaneous triple colour immunofluorescence (phycoerythrin and Cy-Chrome). We were thus able to determine the subpopulations involved in forming conjugates and we found that T-B conjugates were preferentially formed by CD4, CD45RO positive T cells, which are believed to be the memory T lymphocytes.

CD19 regulation of human B cell responses. B cell proliferation and antibody secretion are inhibited or enhanced by ligation of the CD19 surface glycoprotein depending on the stimulating signal used.

The regulation of human B cell proliferation and differentiation by the CD19 surface glycoprotein was investigated. As expected, proliferation induced by costimulation with anti-IgM plus IL-4 or IL-2, or with G28.8 antibody plus IL-4 was inhibited by antibody ligation of CD19. In contrast, proliferation of tonsillar B cells to mitogenic doses of PMA (5 ng/ml) or to EBV were enhanced, and proliferation of B cell lines to BCGF(low) was unaffected. Similarly, specific antibody responses by tonsillar B cells to influenza virus, and Ig secretion by the CESS lymphoblastoid cell line in response to IL-6 were inhibited, whereas polyclonal Ig production in response to EBV was enhanced. These results show that human B cell responses may be inhibited or enhanced by CD19 depending on the stimulating signal used. The difference in response to CD19 ligation did not depend on whether proliferation or differentiation was being measured, or whether stimulation was by surface Ig. In experiments using PMA as a T cell independent mitogen, it was found that ligation of CD19 inhibited proliferation of B cells costimulated with low doses of PMA plus G28.5 (CD40) antibody, but enhanced the response to higher (mitogenic) doses with or without costimulation with G28.5. The change from inhibition to enhancement occurred over a very small increase in PMA dose (0.5-1.0 ng/ml) that corresponded exactly to the lowest dose required for mitogenic activity. Finally, we showed that CD19 ligation inhibited the increase in surface expression of CD23, but not IgM, induced by IL-4, showing that CD19 ligation can have opposed effects on different responses to the same signal. Together our results suggest that CD19 activation of human B cells interacts with other signaling events to enhance or inhibit the subsequent response.

Interleukin-4 stimulates immunoglobulin secretion by Epstein-Barr virus (EBV)-activated tonsillar B cells, and by EBV-transformed lymphoblastoid B cell lines without increasing cell division.

Freshly prepared Epstein-Barr virus-transformed B lymphoblastoid cell lines derived from five different donors were tested for their responses to recombinant human interleukin-4 and to low molecular weight B cell growth factor. In the absence of either cytokine, all five lines secreted immunoglobulin of more than one isotype (IgM, IgG, and IgA, but not IgE). Stimulation with interleukin-4 resulted in a significant increase in immunoglobulin secretion, but did not enhance cell division measured by tritiated-thymidine uptake or cell counts. In contrast, low molecular weight B cell growth factor increased both immunoglobulin secretion and cell division. The increase in immunoglobulin secretion stimulated by interleukin-4 occurred for each of the different isotypes (IgM, IgG and IgA) produced by the unstimulated line. No IgE secretion was detected for any of the five lines. It was also found that low (5 units/ml), but not high (100 units/ml), concentrations of interleukin-4 increased IgM, IgG and IgA secretion by tonsillar B cells polyclonally activated with Epstein-Barr virus. Again, no IgE was detected at any time. These results suggest that interleukin-4 can function as a late-acting B cell differentiation factor as well as a growth factor for human B cells.

Human T cell-replacing factor(s): a comparison of recombinant and purified human B cell growth and differentiation factors.

Conditioned medium from phytohemagglutinin-activated T cells contains T cell-replacing factor(s) (TRF) able to restore specific antibody responses by human blood or tonsillar B cells which have been thoroughly depleted of T cells. Of twelve recombinant cytokines tested as possible candidates for TRF in conditioned media, namely human recombinant interleukin (hrIL) 1 alpha and beta, hrIL2, hrIL3, hrIL4, hrIL5, hrIL6, hrIFN-alpha and -gamma, hr granulocyte macrophage colony-stimulating factor (hrGM-CSF) and tumor necrosis factor (hr TNF)-alpha and -beta only IL2 was found to have TRF activity. In addition, a semi-purified low molecular weight B cell growth factor (BCGFlow) also had TRF activity. As the commercially available BCGFlow is known to contain low concentrations of IL2, IFN-gamma, TNF and GM-CSF as impurities, it was important to exclude these as being responsible for the TRF activity. At the concentrations present in BCGFlow (less than 0.2 U/ml), IL2 was not active in the TRF assay. In contrast, a combination of IL2 (0.2 U/ml), IFN-gamma (50 U/ml), TNF-alpha (50 U/ml) and TNF-beta (100 U/ml) did have TRF activity suggesting that B cells could be made to respond to low doses of IL2 by the presence of other cytokines. Although this finding raises important questions about the nature of TRF in conditioned medium, the TRF activity of BCGFlow was unlikely to be due to such a synergistic combination of cytokines for the following reasons. First, in several experiments, responses were obtained with BCGFlow, but not with IL2 or combinations of IL2 with IFN and TNF. Second, antibody to IL2 was found to inhibit the TRF activity of IL2 but not of BCGFlow. Taken together these findings show that two distinct cytokines (IL2 and BCGFlow) are TRF for human B cells. However, some combinations of cytokines can also have TRF activity underlining the complexities which can arise from working with semi-purified rather than recombinant factors.

Epstein-Barr-virus-transformed lymphoblastoid cell lines derived from patients with X-linked agammaglobulinaemia and Wiskott-Aldrich syndrome: responses to B cell growth and differentiation factors.

Epstein-Barr-virus-transformed B lymphoblastoid cell lines (EBV-transformed LCL) from three patients with X-linked agammaglobulinaemia (XLA), six patients with Wiskott-Aldrich Syndrome (WAS), and seven normal donors, were tested for growth and differentiation in response to human recombinant IL-4, a commercially available, low molecular weight B cell growth factor (BCGFlow), and B cell differentiation factor (BCDF) secreted by the T24 cell line, now known to be IL-6. Proliferation (3H-TdR uptake) by EBV-transformed LCL from both XLA and WAS patients in response to BCGFlow was similar to that obtained with the normal cell lines. In addition, three normal and three WAS, but none of the XLA EBV-transformed LCL, proliferated a little in response to IL-4. All the normal B cell lines secreted IgM, and six out of the seven secreted IgG in response to BCGFlow and BCDF. A similar pattern of response was obtained with the WAS EBV-transformed LCL (6/6 secreted IgM and 4/6 secreted IgG). Several of the normal and WAS EBV-transformed LCL also secreted IgM and IgG in response to IL-4. In contrast, the lines from the XLA patients were abnormal. One secreted large amounts of IgM and two secreted small amounts, but none of the XLA lines secreted IgG constitutively or in response to any of the factors (IL-4, BCDF). The lack of detectable IgG secretion by the XLA lines was probably due to an absence of precommitted IgG B cell precursors transformed by EBV rather than an intrinsic inability to respond to BCGF and BCDF. All of the lines, including those derived from XLA patients, were shown to secrete B cell growth and differentiation factors detected on indicator B cell lines. These results suggest that the abnormal X-linked genes responsible for XLA and WAS do not interfere with B cell responses to B cell growth and differentiation factors.

Increased expression of surface IgM but not IgD or IgG on human B cells in response to IL-4.

Surface IgM (sIgM) was increased up to 10 times on human tonsillar B cells activated with IL-4. No change was observed for surface IgD, IgG or IgE. Other activators of human B cells, such as TPA, EBV and anti-IgM resulted in increased expression of the low-affinity receptor for IgE (CD23), but had no effect on sIgM. IL-4 also increased sIgM expression on prolymphocytic leukaemic (PLL) B cells, whereas TPA significantly reduced the level of sIgM. The effect on sIgM thus seems specific for IL-4, and is consistent with the existence of a unique IL-4-dependent B-cell activation pathway. Preincubation with IL-4 did not 'prime' B cells to proliferate in response to subsequent exposure to anti-IgM, and slightly decreased the response to co-stimulation with IL-4 and anti-IgM. The increase in sIgM expression in response to IL-4, therefore, does not seem to be important for proliferation.

The marmoset B-lymphoblastoid cell line (B95-8) produces and responds to B-cell growth and differentiation factors: role of shed CD23 (sCD23).

The EBV-producing marmoset B-cell line (B95-8), commonly used as a source of EBV for stimulation and transformation of human B cells, was shown to proliferate in response to supernatants containing human B-cell growth factors (BCGF) derived from PHA-activated T cells or the KG-la cell line, and to a commercial low molecular weight BCGF (BCGFlow), but not to recombinant human IL-4 (rhIL-4). In this respect, B95-8 responded in much the same way as human EBV-transformed lymphoblastoid cell lines (LCL). In contrast, B95-8 did not secrete immunoglobulin in response to B-cell differentiation factor (BCDF) containing supernatants from the KG-la cell line, nor to BCGFlow, or IL-6 obtained from the T24 bladder carcinoma cell line, whereas significant responses were obtained with human EBV-transformed LCL. Both B95-8 and control EBV-transformed human LCL secreted BCGF and BCDF detected with the indicator B-cell lines CESS, L4, and HFB1, but only the human LCL secreted BCGF detectable in co-stimulation assays with TPA-activated tonsillar B cells. Unlike EBV-transformed LCL, B95-8 did not express detectable surface CD23, and did not release into the culture medium soluble CD23 (sCD23) recognized by an EIA for the human molecule. Although not releasing detectable sCD23, B95-8 cells did proliferate in response to purified human sCD23, and were found to be 1000 times more sensitive in this assay than EBV-transformed LCL. This may provide a basis for a sensitive bioassay for sCD23. Unlike EBV-transformed LCL, it seems that in vitro proliferation of B95-8 may involve an autocrine loop which does not depend on CD23.

Response of LFA-1-deficient B cells to interleukin 4 (BSF-1) and low molecular weight B cell growth factor (BCGFlow).

T cell-depleted B cells from a patient with LFA-1 deficiency were tested in costimulation assays for responsiveness to recombinant human IL4 (BSF-1) and purified low molecular weight B cell growth factor (BCGFlow). In both cases the response of LFA-1-deficient B cells was comparable with normal controls. Monoclonal antibodies to LFA-1 alpha (CD11a) and beta (CD18) chains were unable to mimic the action of IL4 on normal B cells in costimulation assays with anti-IgM, and did not inhibit normal B cell proliferation in response to IL4 and anti-IgM. Epstein-Barr virus-transformed lymphoblastoid B cell lines (LCL) from normal and LFA-1-deficient donors both responded in proliferation assays to BCGFlow but not IL4. Similarly, both normal and LFA-1-deficient LCL increased IgM secretion in response to BCDF, BCGFlow and, interestingly, IL4. The normal LCL also increased IgG secretion in response to these factors, but no IgG was detected in supernatants from the LFA-1-deficient LCL. These results show that LFA-1 expression is not essential for B cell responses to B cell growth and differentiation factors.

Intestinal hypersensitivity reactions in the rat. I. Uptake of intact protein, permeability to sugars and their correlation with mucosal mast-cell activation.

We have confirmed previous observations that intestinal anaphylaxis induced in rats previously sensitized to ovalbumin (OVA) is associated with an increased uptake of an unrelated 'bystander' protein, bovine serum albumin (BSA) fed 1 hr previously. In this study, this enhanced protein uptake was associated with an increased lactulose/rhamnose excretion ratio after administration of these sugars, although there was no correlation between the two measurements. One hour after antigen challenge the serum levels of rat mast-cell protease II (RMCPII), a specific marker for mucosal mast-cell secretion, were significantly higher than both the pre-challenge levels and those of sham-challenged controls (P less than 0.002). There was a significant positive correlation between the serum levels of RMCPII and the lactulose/rhamnose excretion ratios (P less than 0.05), but no such correlation existed between RMCPII and BSA levels in the challenged rats. In other studies the urinary lactulose/rhamnose ratios of rats with cetrimide-induced gut damage were found to be significantly increased, although BSA uptake into the serum remained unaltered. We conclude that there is no simple correlation between gut permeation of low-molecular weight sugars and and the uptake of macromolecular proteins.

Recombinant human interleukin 5 is an eosinophil differentiation factor but has no activity in standard human B cell growth factor assays.

Following the observation that mouse interleukin 5 (IL5) is active as a B cell growth factor (BCGF) as well as an eosinophil differentiation factor, this work was carried out to test recombinant human IL5 for BCGF activity. A highly active, partially purified batch of recombinant human IL5 was prepared and tested for BCGF activity in four laboratories. This batch gave a 50% endpoint of 1:77,450 in the human eosinophil differentiation assay, 1:983 in the mouse eosinophil differentiation assay and 1:42 in the mouse BCL1 assay, thus demonstrating that, like mouse IL5, human IL5 has cross-species activity. By comparison with the assays in the mouse this batch would be expected to have 50% maximal human BCGF activity of about 1:4000. In each assay a known positive factor was used as a positive control, and there was no inhibitory activity in the preparation. However, despite the activity towards the mouse B cell lymphoma, the results showed no detectable activity in a panel of assays used to identify human BCGF and B cell differentiation factors. These assays included (a) proliferation assays with tonsillar or splenic B cells in the presence of the co-stimulators anti-mu or phorbol myristate acetate; (b) a restimulation assay in which tonsillar B cells are first activated with either Staphylococcus aureus Cowan 1 or a mixture of phorbol dibutyrate and ionomycin, or splenic B cells are first activated with anti-mu; (c) production of immunoglobulin by B cells in a restimulation assay with Staphylococcus aureus Cowan 1; (d) production of immunoglobulin by the Epstein-Barr virus-transformed B lymphoblastoid CESS cell line; (e) the ability to stimulate proliferation of chronic lymphocytic leukemia (B-CLL) cells freshly explanted from three different patients; (f) the ability to stimulate the B lymphoma (L4) cell line and the mature B cell (HBF1) line, and (g) the ability to replace T cells in specific antibody responses. It therefore seems unlikely that recombinant human IL5 is either a growth or a differentiation factor for human B cells, and raises the interesting question of the biological significance of the BCGF activity of this factor in the mouse.

The response of selected human B cell lines to B cell growth and differentiation factors.

Fifteen human B cell lines were tested for their ability to respond to B cell growth and differentiation factors present in phytohemagglutinin-conditioned medium. Five lines responded significantly: CESS showed an increase in IgG production only, HFB1 and BALM1 showed an increase in proliferation only and L4 and BALM4 showed an increase in both IgG production and proliferation. When four of the responding lines (CESS, HFB1, L4 and BALM4) were cultured with human recombinant-derived interleukin 1, interleukin 2, interleukin 4 or interferon-gamma no significant response was seen. CESS, L4 and BALM4 all increased IgG production in response to partially purified B cell growth factor (Cellular Products, Inc., Sera-Lab., Crawley Down, GB) and B cell differentiation factor-containing supernatant from the T24 bladder carcinoma cell line. HFB1, L4 and BALM4 all showed increased tritiated thymidine incorporation in response to purified B cell growth factor but not in response to B cell differentiation factor-containing supernatant. These lines may prove useful in the study of B cell growth and differentiation factors and their receptors.

T cell help in human antigen-specific antibody responses can be replaced by interleukin 2.

Recombinant IL 2, and immunosorbent/high performance liquid chromatography-purified interleukin 2 (IL 2) obtained from the human T cell leukemic line Jurkat, but not interferon-alpha or -gamma, were able to substitute for T cells in specific antibody responses to influenza virus by T cell-depleted (E-) human peripheral blood mononuclear cells, and resulted in antibody formation equivalent to that obtained in the presence of T cells. The antibody response was shown to be antigen specific by using two non-cross-reacting strains of influenza virus (A/X31 and B/HK). IL 2 in this assay therefore functions as a T cell-replacing factor. Less than 1% of T (UCHT1+) cells were present in the E- preparations, and this number did not increase during the 7-day culture with antigen and IL 2. Because the frequency of T helper cells for X31 is known to be less than 5 X 10(-5), this low number of contaminating cells excluded indirect action of IL 2 through antigen-specific T helper cells. Three to four times less IL 2 was required for antibody production by E- cells than was needed for optimal proliferation by an IL 2-dependent T cell line. Moreover, the concentration of anti-Tac required for 50% inhibition of the IL 2-induced antibody response was 50 times less than required for 50% inhibition of IL 2-dependent proliferation by the T cell line. But when T cells were added back to the E- cells, the anti-Tac inhibition curve shifted back to that obtained with the T cell line. In cell labeling experiments, Leu 11+ cells but not HNK1+ cells were increased in E- cells cultured with antigen and IL 2. This increase in Leu 11+ cells was abolished by prior passage of the E- cells through Sephadex G-10 columns without affecting the IL 2-induced antibody response. From these experiments we conclude that IL 2 can replace T cells in specific antibody responses, and that the IL 2 effect is not mediated indirectly through T cells or large granular lymphocytes.

The importance of antibody quality in sandwich ELISA systems. Evaluation of selected commercial reagents.

The detection of low levels of immunoglobulin in cell culture supernatants etc. by ELISA procedures demands high sensitivity and absolute specificity. We have used commercially available antibodies prepared by positive affinity purification and immunoglobulin fractions obtained by ion-exchange chromatography in various combinations in ELISA systems for such quantitative analyses. These demonstrate that when affinity-purified reagents lacking unwanted anti-species cross-reactivity are used as both the capture antibody and the indicator antibody the results are markedly superior (high sensitivity, low backgrounds and a wide working range) compared to those obtained with all other possible combinations.

Appearance of delayed-type hypersensitivity effector cells in murine gut mucosa.

Feeding of a protein antigen, human gamma globulin (HGG), to BALB/c mice prior to parenteral immunization resulted in the abrogation of a delayed-type hypersensitivity (DTH) response to challenge with that antigen. Unlike parenterally immunized mice, HGG-fed mice were unable to transfer DTH to naive syngeneic recipients using peripheral lymph node lymphocytes. Co-transfer experiments ruled out the possibility of a suppressor cell in the orally immunized mice operating on DTH effector cells. Intra-epithelial lymphocytes (IELs) from mice immunized either orally or parenterally were able to transfer a DTH reaction to unimmunized recipients, while mesenteric lymph node lymphocytes from orally, but not parenterally, immunized donors were capable of transferring DTH. The implications of these results for investigations of gastrointestinal disorders with a suspected immunological aetiology are discussed.