T-suppressor cells sensitive to cyclophosphamide and to its in vitro active derivative 4-hydroperoxycyclophosphamide control the mitogenic response of murine splenic B cells to dextran sulfate. A direct proof for different sensitivities of lymphocyte subsets to cyclophosphamide.

As measured by [(3)H]thymidine uptake, spleen cells of mice injected 7 d previously with a single dose of cyclophosphamide (Cy) (125 mg x kg (-1)) gave an enhanced response to dextran sulfate (DS), a diminished response to lipopolysaccharide (LPS), and a normal response to concanavalin A. Addition of syngeneic thymocytes to spleen cells inhibited the enhanced response of the cells to DS and slightly enhanced their response to LPS. Pretreatment of thymocytes by 4-hydroxyperoxycyclophosphamide (4HP-Cy) in vitro (an in vitro active derivative of Cy) abrogated the effect of thymocytes on the DS response but not on the LPS response. Pretreatment of spleen cells by small doses of 4HP-Cy (0.1-1.0 mug. ml(-1)) in vitro enhanced the capacity of the cells to respond to DS but either did not affect, or even diminished their capacity to respond to LPS. The enhancement of the DS response by 4HP-Cy treatment could not be detected using spleen cells depleted of T cells or lacking functioning T cells. 4HP-Cy doses more than 3 mug ml(-1) diminished or abolished the capacity of the spleen cells to respond to LPS as well as their capacity to respond to DS. The results show (a) that in contrast to the LPS-reactive B-lymphocyte subset, the proliferative capacity of DS-reactive subset is negatively controlled by a Cy- and 4HP-Cy-sensitive T-cell subset and (b) that these T- suppressor cells are more sensitive to Cy and 4HP-Cy (to their respective active alkylating metabolites) than B lymphocytes and T cells carrying other immunological functions.

Interleukin 7 induces CD4+ T cell-dependent tumor rejection.

The potential of interleukin 7 (IL-7) to induce an antitumor response in vivo was analyzed. Therefore, the IL-7 gene was expressed in the plasmacytoma cell line J558L. Although the growth of IL-7-producing cells was not retarded in vitro, the IL-7-producing cells were completely rejected upon injection into mice. Tumor rejection was observed only in syngeneic but not in nude mice. The tumor-suppressive effect could be abolished by the parallel injection of an anti-IL-7 monoclonal antibody. Immunohistochemical analysis revealed IL-7-dependent infiltration of the tumor tissue by CD4+ and CD8+ T lymphocytes, and also type 3 complement receptor-positive (CR3+) cells, predominantly macrophages. Depletion of T cell subsets in tumor-bearing mice showed the absolute dependence of the antitumor response on CD4+ cells, whereas tumor rejection was unaffected by depletion of CD8+ cells. In addition to CD4+ cells, CR3+ cells were also needed for tumor rejection. The antitumor effect of IL-7 was confirmed by expression of the IL-7 gene in a second tumor cell line of different cellular origin. Together, our results demonstrate that a high local IL-7 concentration at the tumor site obtained by tumor cell-targeted gene transfer leads to tumor rejection involving a cellular mechanism that seems to be different from the ones observed in analogous experiments with other cytokines.

Analysis of restriction fragment length polymorphism in lymphokine genes of normal and autoimmune mice.

Analysis of RFLP has been employed in lymphokine genes of autoimmune and normal mice. No polymorphism could be detected in the loci containing IL-2, IL-2 receptor, IL-5, and IFN-gamma in NZB, NZW, BxSB, and MRL/lpr mice when compared with normal mice. Allelic forms were identified in the IL-1 alpha gene of BALB/c and in the IL-4 gene of NZW. The frequency of the Bam HI RFLP in the TNF-alpha gene of NZW which has been proposed to be associated with the development of autoimmune disease in (NZB x NZW)F1 mice has been analyzed in a number of different inbred strains and in wild mice. Since the same allele is inherited in most autoimmune, healthy laboratory and wild mice the TNF-alpha gene does not seem to be one of the causal agents that contributes to the development of autoimmunity in (NZB x NZW)F1 mice.

DNA rearrangement and constitutive expression of the interleukin 6 gene in a mouse plasmacytoma.

To study the potential involvement of IL-6 in the development of plasmacytomas, a number of plasmacytoma lines were analyzed for alterations in the IL-6 locus. A DNA rearrangement due to the insertion of an intracisternal A particle retrotransposon 18 bp 5' of the transcriptional start site was detected in the cell line MPC11. The IL-6 gene is constitutively expressed in MPC11, suggesting the involvement of IL-6 in the development of certain myeloma/plasmacytomas according to the "autocrine growth hypothesis".

B cell-stimulatory factor 1 (BSF-1) promotes growth of helper T cell lines.

T cell-derived supernatants (SN) that contain B cell-stimulatory factor 1 (BSF-1) and lack IL-2 promote the growth of the IL-2-dependent T cell line, HT-2, as well as three other clones or lines of T cells that can provide help to B cells. The BSF-1 purified from these SNs promotes growth of HT-2 cells approximately 50% as effectively as purified IL-2. A potential involvement for contaminating IL-2 in the BSF-1 preparations was excluded by the demonstration that anti-BSF-1 mAbs blocked the BSF-1-induced growth of HT-2 cells; in contrast, these antibodies did not block the IL-2-induced proliferation of the HT-2 cells. In addition, anti-IL-2 mAbs or anti-IL-2-R antibodies blocked the HT-2 growth-promoting activity of purified IL-2, but not BSF-1. Finally, BSF-1 promoted only a very modest growth of Con A-induced T cell blasts, and failed to induce significant growth in seven other cytotoxic, alloreactive, and long-term T cell lines. Taken together, these results indicate that in addition to its known effects on resting and LPS-stimulated B cells, BSF-1 can promote growth of certain subsets of activated T cells, in particular, those that provide help to B cells.

Expression of tumor necrosis factor by different tumor cell lines results either in tumor suppression or augmented metastasis.

Tumor necrosis factor (TNF) produced by tumor cells after gene transfer can effectively suppress the growth of locally growing tumors. We wanted to test the effects of "local" TNF on the growth of a highly metastatic cell line. Therefore, a recombinant retrovirus allowing expression of the TNF gene by the beta-actin promotor has been constructed and used to infect the two tumor cell lines EB and ESB, which grow as solid tumor or metastasize, respectively. Expression of TNF by EB cells resulted in their rapid and dose-dependent rejection. In sharp contrast, mice injected with ESB cells producing similar amounts of TNF showed no signs of tumor suppression, but rather had reduced survival rates that correlated with enhanced hepatic metastases. The accelerated formation of liver metastases by ESB TNF cells could be reversed by an anti-TNF mAb. These results demonstrate the opposite effects TNF may have on tumor growth: suppression of a locally growing tumor and promotion of metastasis formation.

Administration of group A streptococcal cell walls to rats induces an interleukin 2 deficiency.

Intraperitoneal administration of group A streptococcal cell walls to Lewis rats induces a chronic arthritis, whereas the Fischer strain is resistant to the development of the lesion. Spleen cells from cell wall-treated rats (Lewis and Fischer) are deficient in the synthesis of IL-2. Using an mAb directed against the rat IL-2-R, the present studies indicate that the expression of IL-2-R on spleens of cell wall-treated rats is normal. However, the addition of exogenous IL-2 to spleen cells cultured with Con A does not stimulate the mitogenic response.

Anti-interleukin 2 receptor monoclonal antibodies spare phenotypically distinct T suppressor cells in vivo and exert synergistic biological effects.

The therapeutic efficacies of ART-18, ART-65, and OX-39, mouse antibodies of IgG1 isotype recognizing distinct epitopes of the p55 beta chain of the rat IL-2-R molecule, were probed in LEW rat recipients of (LEW X BN)F1 heterotopic cardiac allografts (acute rejection in untreated hosts occurs within 8 d). A 10-d course with ART-18 prolongs graft survival to approximately 21 d (p less than 0.001). Therapy with ART-65, but not with OX-39, was effective (graft survival approximately 16 and 8 d, respectively). Anti-IL-2-R mAb treatment selectively spared T cells with donor-specific suppressor functions; the CD8+ (OX8+ W3/25-) fraction from ART-18-modified recipients, and primarily the CD4+ (W3/25+ OX8-) subset from ART-65-treated hosts conferred unresponsiveness to naive syngeneic rats after adoptive transfer, increasing test graft survival to approximately 16 and 45 d, respectively. Concomitant administration of ART-18 and ART-65 to recipient animals in relatively low doses exerted a strikingly synergistic effect, with 30% of the transplants surviving indefinitely and 50% undergoing late rejection over 50 d. These studies provide evidence that anti-IL-2-R mAbs selectively spare phenotypically distinct T cells with suppressor functions. The data also suggest that in vivo targeting of functionally different IL-2-R epitopes may produce synergistic biological effects.

Tumor suppression after tumor cell-targeted tumor necrosis factor alpha gene transfer.

The tumor necrosis factor alpha (TNF-alpha) gene was introduced by retroviral gene transfer into the TNF-alpha-insensitive tumor cell line J558L. Production of 40 pg/ml TNF-alpha by clone J2T12 consistently did not change the growth rate in vitro, but drastically suppressed tumor growth when injected into syngeneic BALB/c mice. Within 2 wk, 90% of the mice inoculated with J558L cells developed a tumor, but none of the mice injected with J2T12 did so. Within the observation period (greater than 3 mo), 60% of the mice inoculated with J2T12 did not develop a tumor. In the other 40% of the mice, tumor manifestation was significantly delayed. Mice injected simultaneously with J2T12 cells and an anti-TNF-alpha monoclonal antibody developed tumors similar to parental J558L cells. Similarly, the tumor-suppressive effects of TNF-alpha were abolished, e.g., by injection of an anti-type 3 complement receptor (CR3) monoclonal antibody that is known to prevent migration of inflammatory cells. These results and the observation of tumor-infiltrating macrophages suggest that lack of tumorigenicity of J2T12 cells is due to the TNF-alpha secretion by the tumor cells and that TNF-alpha acts indirectly by a mechanism that involves chemotactic recruitment and activation of cells, predominantly of macrophages. In contrast, the tumor growth was not affected when, instead of TNF-alpha, interleukin 6 was expressed by J558L cells. Together, our results support the concept of tumor cell-targeted cytokine gene transfer as a tool for cancer treatment, and particularly demonstrate that extremely low doses of TNF-alpha produced by tumor cells are sufficient to inhibit tumor growth without detectable side effects.

Suppression of cell-mediated and humoral immune responses by an interleukin-2-immunoglobulin fusion protein in mice.

Interleukin-2 (IL-2) plays a pivotal role in the cellular and humoral immune responses directed against foreign antigens. We characterized the in vitro and in vivo properties of a chimeric protein consisting of mouse IL-2 fused to the mouse IgG2b Fc domains. This fusion protein binds to IL-2 and Fc receptors and supports IL-2-dependent cell proliferation but does not mediate lysis of IL-2 receptor-positive cells in the presence of murine complement in vitro. However, in vivo the IL2-IgG2b fusion protein suppresses both cellular and humoral immune responses after immunization with sheep erythrocytes. Surprisingly, delayed hypersensitivity is inhibited despite a dramatic increase of splenic CD3+ and NK1.1+ lymphocytes, indicating that altered homing of IL2-IgG2b-activated lymphocytes rather than cytolysis prevents these cells from accumulating in areas of inflammation. Although in vitro the IL2-IgG2b fusion protein does not alter proliferation of B cells in response to mitogenic stimulation, IgM production in response to sheep erythrocytes is profoundly inhibited in mice treated with the IL2-IgG2b fusion protein. Since no side effects are observed, the IL2-IgG2b fusion protein may expand the therapeutic repertoire of reagents used for the treatment of allograft rejection and autoimmune diseases.

Interleukin 10 transfected into Chinese hamster ovary cells prevents tumor growth and macrophage infiltration.

Expression of cytokines in tumor cells provides a sensitive modality to analyze the consequences of local cytokines in vivo on tumor infiltrating cells and tumorigenicity. We have transfected Chinese hamster ovary (CHO) cells with an interleukin 10 (IL-10) expression vector. CHO-IL10 cells although unaltered with respect to their in vitro growth lost tumorigenicity, both in nude and in SCID mice and in an IL-10 dose dependent manner. In addition, CHO-IL10 cells suppressed the growth of equal numbers of coinjected but not of contralaterally injected CHO cells. Immunohistology with anti-CR3/Mac-1 and anti-Mac-3 monoclonal antibodies revealed that CHO tumors were substantially infiltrated by macrophages. However, in CHO-IL10 tumors macrophages were virtually absent within the tumor tissue. Our results suggest that IL-10 indirectly suppresses tumor growth of certain tumors by inhibiting infiltration of macrophages which may provide tumor growth promoting activity.

Vaccinations with tumor cells genetically engineered to produce different cytokines: effectivity not superior to a classical adjuvant.

The potential of tumor cells (J558L) engineered to produce one of 5 different cytokines (interleukin 2, interleukin 4, interleukin 7, tumor necrosis factor, or gamma-interferon) to give rise to systemic immunity protective against a contralateral challenge with the parental cells was analyzed. The rejection of all cytokine-producing cells appeared to induce some systemic response capable of mediating the rejection of low numbers of subsequently contralaterally injected cells, but the effect was much less obvious with higher cell numbers. The injection of any possible combination of two of the cytokine producers did not reveal any synergistic effects. The cytokine gene-transfected tumor cells were not superior to the parental cells admixed with the adjuvant Corynebacterium parvum with respect to their potential as immunogens to induce immunity.

A sialyl-Le(x)-negative melanoma cell line binds to E-selectin but not to P-selectin.

The adhesion molecules E-selectin (ELAM-1) and P-selectin (GMP-140/CD62) recognize the carbohydrate motives sialyl-Le(x), sialyl-diLe(x), or sialyl-Lea, though with different affinity. We found that the melanoma cell line NKI-4 bound to E-selectin, but not to P-selectin. This melanoma cell line did not express sialyl-Le(x), but was positive for sialyl-diLe(x) and sialyl-Le(a). In contrast, 2 other melanoma cell lines, MeWo and SK-MEL-28, expressing either sialyl-diLe(x) or sialyl-Le(a) on the cell surface, bound neither E-selectin nor P-selectin. Transfection of the fucosyltransferases Fuc-TIII, Fuc-TIV, and Fuc-TV mediates cell surface expression of sialyl-Le(x) in many cell lines. We detected transcripts of the fucosyltransferases Fuc-TIII and Fuc-TV in 4 melanoma cell lines despite the absence of cell surface sialyl-Le(x). Our observations indicate that expression of fucosyltransferases (Fuc-TIII and -TV) and generation of cell-surface sialyl-diLe(x) are not sufficient to permit adherence to E-selectin or P-selectin. Furthermore, it seems possible that a yet undefined ligand different from sialyl-Le(x), sialyl-diLe(x), or sialyl-Le(a) enables melanoma cells to adhere to E-selectin.

The high affinity interleukin 2 receptor: evidence for three distinct polypeptide chains comprising the high affinity interleukin 2 receptor.

Low and high affinity receptors for interleukin 2 were investigated on interleukin 2 (IL-2) receptor bearing cells by chemical cross-linking of 125I-labelled IL-2 to its receptor, or membrane proteins associated with the IL-2 binding sites. SDS-PAGE analysis of the cross-linked complexes of the murine CTLL 16 cells and human T-blasts, which bear high and low affinity IL-2 receptors, showed three distinct bands. The fastest of those three bands ran in parallel to the single band of 65-70 kDa found on the only low affinity receptor bearing mouse T-lymphoma Eb, which is thought to be one beta-chain (55 kDa IL-2 binding protein) and one IL-2. Both upper bands ran in parallel with those produced by the 2C8 clone of the NK-like cell line YT which lacks the 55 kDa binding protein and bears only a single class of receptors with an intermediate affinity. Internalisation studies using CTLL 16 cells revealed that all three bands disappeared under conditions allowing receptor internalisation. Low and high affinity binding sites of CTLL 16 cells were destroyed by trypsinisation and the IL-2 binding properties of the cells were regenerated in parallel with the reappearance of all bands. These results show in addition to the beta-chain (55 kDa binding protein) and the alpha-chain 75 kDa binding protein, an IL-2 membrane protein complex with an apparent mol. wt of 115 kDa in CTLL 16 cells. They are the first direct indication of a putative gamma-chain of the high affinity IL-2 receptor.

Studies on the interleukin-2 receptor, its generation and dynamics using monoclonal anti-interleukin-2 receptor antibodies.

There is increasing evidence suggesting that the monoclonal antibodies ART-18, AMT-13 and anti-Tac recognize species-specific antigenic determinants of the interleukin-2 (IL-2) receptors of rat, mouse and human origin, respectively. In order to compare directly the molecules (glycoproteins) recognized by these antibodies, concanavalin A (ConA) activated T-lymphocytes of the respective species were surface labeled with 125I, after which the materials immunoprecipitated by the appropriate anti-IL-2 receptor antibodies were subjected to SDS-PAGE analysis. The noncross-reacting antibodies ART-18 and AMT-13 both precipitated a 50-55-kD molecule. The anti-Tac-reactive material (the putative human IL-2 receptor) is considerably different (60-65 kD) from those precipitated by antibodies ART-18 and AMT-13 (the putative rat and mouse IL-2 receptors). An indirect binding assay using the anti-mouse IL-2 receptor antibody AMT-13 showed that, after addition of ConA to spleen cell cultures, T-lymphocytes expressed IL-2 receptors before the onset of the ConA-induced DNA synthesis. The ConA-induced expression of the IL-2 receptor is apparently a transient event. IL-2 receptor bearing cells progressively lost their receptors (within 6 days) when recultured in the absence of ConA. Cells re-exposed to ConA regained IL-2 receptors. Short exposure of T-cells (thymocytes) to ConA or the nonmitogenic compound phorbol myristate acetate (PMA) is not sufficient to trigger IL-2 receptor expression. Murine thymocytes incubated with PMA for 30 min or with ConA for 4 hr (mitogen-pulsed T-cells) failed to bind the anti-IL-2 receptor antibody AMT-13 and to absorb IL-2 activity present in semipurified IL-2 preparations, but they proliferated vigorously in response to the same IL-2 preparations. The IL-2 preparations, when absorbed with thymocytes, lost: (1) the capacity to generate IL-2 receptors, and (2) the capacity to induce proliferation of mitogen-pulsed cells; but they retained the capacity to induce proliferation of T-lymphoblasts. These results suggest the existence of a factor, IL-2 receptor inducing factor (RIF), present in the IL-2 preparations. It is postulated that RIF is a prerequisite for the acquisition of IL-2 receptors and consequently for IL-2 responsiveness by lectin-activated cells.

Lack of tumorigenicity of interleukin 4 autocrine growing cells seems related to the anti-tumor function of interleukin 4.

Recently, the failure of interleukin 4 (IL4) autocrine growing CT4S cells to grow in vivo has been demonstrated. Because it could not be excluded that the cells produce insufficient amounts of IL4 to support their growth in vivo, subclones were established which are unresponsive to exogenous IL4 and therefore have acquired full growth autonomy. From the fact that the subclones likewise did not give rise to tumors when injected into nude mice, one may conclude that the IL4 production of autocrine growing CT4S prevents their growth in vivo. To test this hypothesis, a retroviral vector containing the IL4 gene under the control of the immunoglobulin heavy chain (Igh) enhancer/promoter was constructed and used to infect the myeloma cell line J558L. An IL4 producing clone was established (J558L-XEPIL4) and the tumor progression in comparison to the parental clone J558L was monitored in nude mice. The IL4 production significantly delayed the growth of J558L-XEPIL4 in vivo. Tumor suppression was much more evident when J558L-XEPIL4 cells were injected into syngeneic BALB/c mice. These results may explain why autocrine growing CT4S do not grow in vivo and suggest the involvement of functional T lymphocytes in the effectiveness of the host dependent anti-tumor action of IL4.

Structure and evolution of mouse interleukin 6 gene.

Restriction fragment length polymorphism in the interleukin 6 gene of murine rodents extending phylogenetically from Mus musculus domesticus to the rat has been analyzed. Most species exhibit distinct restriction site patterns. In contrast, limited polymorphism was found in the tumor necrosis factor alpha gene indicating different selective pressure acting on both genes. The gene encoding interleukin 6 was isolated from a genomic library and the exon/intron organization was determined by restriction analysis and limited DNA sequence analysis. It consists of five exons which distribute over about seven kilobases, thus resembling in structure and organization the human counterpart. Furthermore, no restriction fragment length polymorphisms in the interleukin 6 gene of autoimmune strains NZB, NZW, MRL-lpr/lpr and BxSB could be detected for either EcoRI, BamHI or HindIII.

Regulation of interleukin-2 receptor expression and receptor release.

The generation and cell surface expression of IL-2 receptors was monitored by: (i) an ELISA that permits quantitative determination of detergent-solubilized or soluble IL-2 receptors; and (ii) detection of the binding of 125I-labelled recombinant IL-2 and of anti-IL-2 receptor antibodies to receptor bearing cells. Upon lectin stimulation both high and low affinity IL-2 receptors became expressed in parallel at the cell surface. Both high and low affinity receptors were upregulated by IL-2. Upon lectin activation the amount of cell-associated receptors increased and on day 2 of the culture period IL-2 receptors were detectable in the culture supernatant. IL-2 upregulated both high and low affinity IL-2R expression on T-lymphoblasts. IL-2R bearing leukemic cells and T lymphoblasts released IL-2R when cultured in vitro. IL-2R release by T lymphoblasts was enhanced dramatically by IL-2. On the other hand, IL-2-receptor positive leukemic cells released receptors in an IL-2 independent manner. Release of receptors could also be detected in serum-free medium. At least a part of the released receptors could be specifically bound to immobilized pure recombinant IL-2 and to monoclonal anti-IL-2-receptor antibodies. Small but significant amounts of soluble IL-2 receptors were detectable in the sera of normal mice. In sera of mice inoculated with IL-2-receptor positive syngeneic leukemic cells, elevated levels of IL-2 receptors were detectable. Release of IL-2 receptors seems to represent one of the major routes by which the receptors are cleared from the cell surface.

A general method for chimerization of monoclonal antibodies by inverse polymerase chain reaction which conserves authentic N-terminal sequences.

Chimerization of antibodies (Ab) by cloning the V (variable) regions encoding the light and heavy chains with degenerate oligodeoxyribonucleotide primers matching to framework region 1 and to the joining regions, leads to Ab with altered amino acids at the N-terminus compared to those of the parental Ab. This is due to N-terminal framework 1 sequences in the expression vectors [Larrick et al., Bio/Technology 7 (1989) 937-938; Le Boeuf et al., Gene (1989) 371-377; Orlandi et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837]. This might lead to Ab with altered affinity to the antigen due to interaction of framework sequences with complementarity determining regions. Moreover, some V regions may be refractory to cloning by this procedure. Here, we describe a method to circumvent these potential problems. The V regions for both chains of the Ab are cloned by inverse polymerase chain reaction (PCR) with primers matching the known constant region sequences of the Ab. After sequencing, PCR fragments corresponding to the V regions of both chains are inserted in-frame into appropriate expression vectors leading to Ab with unaltered N-terminal sequences after expression in mammalian cells. The procedure is illustrated with an Ab directed against the beta chain of the human interleukin-2 receptor.

Identification of the blue-sensitive cones in the mammalian retina by anti-visual pigment antibody.

Monoclonal antibodies to visual pigments produced in our laboratory were applied to analyze the distribution of color-specific photoreceptor cells in the retina (photoreceptor mosaic). We demonstrated in two ways that the monoclonal antibody OS-2 specifically recognized the blue-sensitive cone cells in the mammalian retina. First, rabbit photoreceptors damaged selectively by intense blue light were recognized by OS-2 antibody. Second, OS-2-positive cones in the ground squirrel were those with thick inner segments, which is known to be characteristic of the blue-sensitive cones. In addition, the OS-2-positive cones in monkeys have a distribution and pattern characteristic of blue-sensitive cones in that species. In several other species (human, rabbit, cow, and pig), the OS-2-positive cones represent an appropriate minority of the population of photoreceptor cells. The visual pigment recognized by the OS-2 antibody had a relative molecular weight of 36,000, as shown by immunoblotting of 3 mammalian species. All other cones were recognized by another monoclonal antibody, COS-1, which is regarded as specific to middle-to-long-wavelength-sensitive photoreceptors.