Addition Of Recombinant Interleukin Research Articles

Direct killing of xenograft cells by CD8+ T cells of discordant xenograft recipients.

Long-term pig xenografts in monkeys demonstrated the infiltration of CD8 T cells into pig cartilage xenografts, transplanted into monkeys. The objective of the present study was to determine in an experimental animal model whether CD8 T cells in pig xenograft recipients exert any direct cytotoxic effect on pig cells. The killing of xenograft cells by CD8 T cells, obtained from xenograft recipients, was studied in alpha1,3galactosyltransferase knockout mice that were repeatedly injected intraperitoneally with pig kidney membranes. The pig kidney cell line PK15, which shares many antigens with pig kidney membranes, served as a model for xenograft target cells in cytotoxicity assays. Cell lines from other species were also studied as target cells. Lymphocytes obtained freshly from spleens of mice immunized with pig kidney membranes failed to display significant cytotoxic activity against pig cells. However, incubation of these lymphocytes with irradiated PK15 cells and addition of recombinant interleukin (IL)-2 (100 U/mL), on the third day of incubation, resulted in extensive proliferation and expansion of CD8 cytotoxic T lymphocytes (CTL). These CTL, obtained after 12 days of incubation, killed nonspecifically pig, human, and mouse normal and malignant cells. These CTL were not generated in cultures in the absence of stimulatory pig cells or in the absence of IL-2. These CTL could not be generated in cultures of lymphocytes from naive mice that were incubated with PK15 cells and IL-2. The data obtained imply that CD8 T cells from xenograft recipients can be stimulated in vitro by xenoantigens and IL-2 to differentiate into highly reactive nonspecific CTL that are capable of killing a large variety of xenogeneic and syngeneic cells. Similar in vivo microenvironmental conditions within the xenograft may induce the local differentiation of infiltrating CD8 T cells into CTL that can destroy nonspecifically adjacent xenograft cells. Such cells may not be active outside the xenograft because of the absence of IL-2 in sufficiently high concentrations.

Very low density lipoproteins and interleukin 2 enhance the immunogenicity of 9- O-acetyl-GD3 ganglioside in BALB/c mice

Gangliosides expressed by tumor cells constitute potential targets for immunotherapy. A major limitation of protocols aiming to immunize patients against tumor gangliosides is the weak immunogenicity of these molecules. We have previously shown that exogenous gangliosides are essentially bound to serum lipoproteins. In this study we have analyzed the influence of human serum lipoproteins on the immunogenicity of purified human ganglioside 9- O-acetyl-GD3 in BALB/c mice. Although expressed at very low levels in mice, this ganglioside was not immunogenic when administered in the form of micelles. However 9- O-acetyl-GD3 adsorbed onto Very Low Density Lipoproteins (VLDL) was strongly and reproducibly immunogenic, inducing both an IgM and an IgG response, with higher titers than those obtained with total serum. The IgM antibody response appeared after a single injection whereas the IgG response was observed after 3 weeks but was stronger and more durable. The antibody response to 9- O-acetyl-GD3 bound to other serum fractions was weak or absent. The addition of recombinant interleukin 2 (IL-2) enhanced weak antibody responses to 9- O-acetyl-GD3 thereby facilitating responses to ganglioside in micelles and in protein-free Very Low Density Particles. Using in vitro assays, we demonstrated that VLDL-bound ganglioside 14 C -GM3 was more sensitive to the effect of neuraminidase than gangliosides bound to other lipoprotein fractions, suggesting greater accessibility of VLDL-bound gangliosides. These results indicate that VLDL-bound gangliosides are the most immunologically active fraction of serum gangliosides. VLDL or similar particles and recombinant IL-2 may be useful adjuvants for immunization with gangliosides.

Impaired natural killer cytotoxicity in peripheral blood mononuclear cells in Graves' disease.

We studied the natural killer (NK) activity of peripheral blood mononuclear cells (PBMC) in patients with Graves' disease (GD). Peripheral blood mononuclear cells from 20 untreated hyperthyroid patients with GD showed a significantly reduced NK activity against 51 Cr-labeled K562 cells (33.9 +/- 15.9%), while in 32 euthyroid patients under antithyroid drug therapy. NK activity was similar to that of controls (46.9 +/- 17.3 and 49.9 +/- 20.2%, respectively). Furthermore, normalization of thyroid function with antithyroid drugs was associated with a significant increase and normalization of NK activity during the follow-up of nine GD patients (from 29.2 +/- 17.9 to 48.1 +/- 16.5%). This phenomenon could not be ascribed to a defective number of NK cells because the amounts of CD56+ and CD16+ cells in PBMC from both hyperthyroid and euthyroid GD patients were within normal ranges. Natural killer activity of PBMC from patients with toxic multinodular goiter was similar to that of normal controls (45 +/- 12.8 to 49.9 +/- 20%). No correlation was found between natural killer activity and serum levels of free thyroxine, TSH-inhibitory immunoglobulins, thyroidal antibodies to thryoglobulin and thyroidal microsomal antigen, dose or duration of antithyroid drug therapy. Natural killer activity from both controls and GD patients was enhanced in vitro by addition of recombinant interleukin 2 (IL-2), reaching control levels in hyperthyroid patients. These abnormalities were not associated with a defective IL-2 production by T cells, nor with a decreased IL-2R expression. We conclude that in untreated Graves' disease there is a decrease in NK cell activity in PBMC, probably dependent on the autoimmune process.(ABSTRACT TRUNCATED AT 250 WORDS)

Transfection of transforming growth factor-beta producing tumor EMT6 with interleukin-2 elicits tumor rejection and tumor reactive cytotoxic T-lymphocytes.

One mechanism by which potentially immunogenic tumors evade the immune response is production of immunosuppressive factors. The murine mammary sarcoma EMT6 has previously been demonstrated to inhibit the proliferation of B-cells, suggesting that this tumor produces immunosuppressive factors. Here, we show that supernatant from EMT6 inhibits the development of cytotoxic T-lymphocytes (CTLs) and that this inhibition can be reversed by addition of recombinant interleukin (IL)-2. Furthermore, we show that EMT6 produces high levels of the immunosuppressant factor transforming growth factor (TGF)-beta. To determine if the T-cell growth factor IL-2 within the tumor microenvironment could reverse the immunosuppressive effect of EMT6, we transfected EMT6 with an expression vector containing the cDNA for murine IL-2 under the control of the beta-actin promoter. These transfectants produce significant levels of IL-2 (26 U/ml). EMT6/IL-2 is rejected by mice at 100-fold higher challenge than are parental cells or control transfectants (neomycin resistance only). Thy-1-expressing cells purified from EMT6/IL-2 tumors show greater cytotoxicity against the parental EMT6 cells than do those from the control transfectant. Thus, IL-2 can reverse the effects of TGF-beta on development and/or proliferation of CTL reactive with EMT6, allowing the establishment of mature effectors in vivo. This has significant implications for the development of CTL and immunotherapy for immunosuppressive tumors.

Murine T-lymphocyte proliferation induced by interleukin 2 correlates with a transient increase in p56lck kinase activity and the tyrosine phosphorylation of a 97-kDa protein.

The addition of recombinant interleukin 2 (rIL-2) to anti-CD3-activated murine G0 phase T cells results in an increased level of tyrosine phosphorylation of a single 97-kDa protein. The degree of tyrosine phosphorylation paralleled the amount of rIL-2 added and correlated with the extent of DNA synthesis. IL-2 treatment resulted in a transient increase in p56lck kinase activity without detectable modification of its level of tyrosine phosphorylation and gel mobility. When G0 T cells were activated by phorbol dibutyrate in the absence of IL-2, the high-affinity IL-2 receptor (IL-2R) expressed failed to induce a proliferative signal, and neither the tyrosine phosphorylation of the 97-kDa protein nor the transient increase in p56lck kinase activity was detected. Northern analysis of the total RNA extracted from these cells showed the accumulation of IL-2R alpha chain-specific mRNA but neither c-myc nor cdc2 mRNA was expressed. The addition of 100 nM rIL-2 to T cells activated by phorbol dibutyrate was able to induce a proliferative response, and under these conditions tyrosine phosphorylation of the 97-kDa protein, the transient increase in p56lck kinase activity, and specific mRNA for IL-2R alpha chain, c-myc, and cdc2 were detected. Unstimulated G0 T cells responded to 100 nM rIL-2 in the same manner as phorbol dibutyrate-activated cells. Irrespective of the signal-transducing structures involved, the IL-2-induced proliferative response closely correlates with an increase in p56lck kinase activity along with the tyrosine phosphorylation of a 97-kDa protein.

A murine thymic stromal cell line which may support the differentiation of CD4 −8 − thymocytes into CD4 +8 − αβ T cell receptor positive T cells

A fibroblastoid cell line TSt-4 was established from fetal thymus tissue of C57BL/6 mice. When fetal thymus (FT) cells or CD4 −8 − (DN) cells of adult thymuses were cultured on the monolayer of TSt-4, a considerable proportion of lymphocytes expressed CD4 or both CD4 and CD8 within 1 day, and the CD4 +CD8 − cells were maintained further while the CD4 +8 + cells disappeared by Day 5. A large proportion of cells generated from DN cells but not FT cells was shown to express CD3 and T cell receptor αβ. Addition of recombinant interleukin (IL)-7 into the cultures resulted in a marked increase of cell recovery without virtual change in differentiation process of αβ lineage. The present work strongly suggests that thymic fibroblasts play an important role in T cell differentiation and IL-7 contributes to supporting proliferation of differentiated cells.

Cellular response and anti-HBs synthesis in vitro after vaccination with yeast-derived recombinant hepatitis vaccine

Two groups of subjects receiving two different doses of yeast-derived recombinant hepatitis B surface antigen (rHBsAg) (10 μg Gen-HB-Vax, Merck Sharp and Dohme and 20 μg Engerix-B, Smith Kline and French) were investigated for in vitro specific humoral and cellular response to the native protein. In vitro proliferative response was dependent on the following critical variables: (1) antigen-specific precursor lymphocytes were present in the peripheral blood for a very short time; (2) the number of circulating specific precursors was dependent on the dose of HBsAg used for vaccination; (3) the presence of antigen-presenting cells was necessary to obtain a blastogenic response in vitro. In vitro proliferation was enhanced by the addition of recombinant interleukin 2 (rIL-2). Spontaneous and stimulated (anti-CD3, pokeweed mitogen) anti-HBs antibody production in vitro was obtained in only eight out of 20 subjects after the fourth boost. Although a different immunogenicity of the two vaccines cannot be excluded, these data strongly suggest that T and B cells responsive to HBsAg present different kinetics of recirculation in the peripheral blood, depending on the antigen dose used for immunization.

Interleukin 4 induces CD8α expression on T cell receptor Vγ5 thymocytes

It is generally accepted that most T cell receptor (TcR) gamma/delta cells are CD4-CD8-. After in vitro culture; however, a low percentage of these cells express the CD8 alpha subunit. We show here that addition of recombinant interleukin (IL) 4 to IL 2-cultured murine TcR V gamma 5 thymocytes induces the expression of CD8 alpha; CD8 beta is not expressed. Co-addition of the anti-IL 4 mAb 11B11 abrogates the induction of CD8 alpha expression, ruling out the possibility of a contaminant. Furthermore, we demonstrate that a substantial part of freshly prepared TcR V gamma 5 thymocytes express CD8 alpha.

Spontaneous and cytokine‐inducible ‘natural’ immunoglobulin secreting cells in organized lymphoid tissues of mice

The number and frequency of spontaneous and cytokine-inducible 'natural' immunoglobulin-secreting cells (ISC) were determined in bone marrow (BM), spleen and Peyer's patch (PP), in vitro. Cells were cultured at limiting dilution in the presence or absence of exogenous recombinant cytokines and supernatants then assayed for total immunoglobulin (Ig) and Ig isotype using an ELISA. Most spontaneous ISC were found in the spleen and BM, with fewer in PP. The addition of recombinant interleukin 5 (rIL-5) promoted a marked increase in both the ISC frequency and the amount of Ig secreted/ISC whereas recombinant IL-6 (rIL-6) promoted only a marginal increase. Recombinant IL-4 (rIL-4) promoted a marginal increase in ISC frequency only. The isotype profile of ISC was in the order IgM greater than IgG2 greater than IgA greater than IgG3 greater than IgG1. The exposure of cells to 1200 rad of gamma-radiation resulted in decreased numbers of spontaneous ISC in all tissues, but the addition of rIL-5 or rIL-6 to the irradiated cells increased both the ISC frequency and Ig secreted. The Ig isotype profile was similar to that of non-irradiated ISC with a few minor exceptions. This large population of potential cytokine-inducible ISC could contribute to 'natural' Ig secretion in vivo.

Anti-CD4 mediates clonal anergy during transplantation tolerance induction.

Depletion of CD4+ cells using anti-CD4 monoclonal antibodies leads to allograft tolerance. Here we show that anti-CD4-mediated tolerance to pancreatic islets of Langerhans transplanted from an A/J (IEk) donor to a diabetic C57B1/6 (B6) (IE-) recipient occurs in the absence of clonal deletion of the potentially IE-reactive V beta 11+ T cells. Instead, a state of clonal anergy is induced in both the CD4+V beta 11+ and CD8+V beta 11+ T cell subsets. This clonal anergy can be partially overcome in vitro by the addition of recombinant interleukin 2.

Interleukin-1 corrects the defective autologous mixed lymphocyte response in multiple sclerosis

Patients with chronic progressive multiple sclerosis (MS) have alterations of T cell regulation that can be measured by in vitro assays and include decreases of the autologous mixed lymphocyte reaction (AMLR). Whether a defect in cytokine secretion was involved in the altered AMLR was investigated in 29 MS patients and 13 age- and sex-matched controls. The response of CD4 + T cell populations to irradiated non-T cells was decreased in MS as compared to control subjects. As previously reported, decreases in the AMLR were similarly observed with whole T cells of MS subjects as compared to controls. The addition of recombinant interleukin (IL)-1 to cultures of either whole T cells or CD4 + T cells with irradiated non-T cells in the AMLR corrected the immune defect in subjects with MS but had no effect on the AMLR in control subjects. In contrast, addition of rIL-2 or rIL-4 to the AMLR did not correct the decreased AMLR in MS patients as compared to controls. The lymphokines IFN-γ and TGF-β2 both decreased the AMLR in MS patients and controls while TNF had no effect. Further, the magnitude of the AMLR response corresponded to IL-1 secretion induced by LPS in the non-T cell population. These studies indicate that defects in IL-1 may be related to immune defects of suppression in MS patients. Selective correction of immunoregulatory defects using lymphokines or their inducers in subjects with autoimmune diseases such as MS may be possible.

Expression of the Alpha‐Chain Gene in Heterogeneous IgA Immunodeficiency

Five heterogeneous IgA-immunodeficient patients were analysed for expression of the alpha-chain gene. The number of surface IgA-bearing B cells was low in four patients. Southern blot analysis indicated no deletion of immunoglobulin structural genes coding for C alpha or alpha switching-region genes. The number of surface IgM and IgA double-bearing B cells increased in some patients. Addition of recombinant interleukin 4 (rIL-4), rIL-5, and rIL-6 to the normal B cells enhanced IgA production. However, B cells of the patients showed no or one-third lower IgA production in response to these lymphokines, even though there was proliferation. rIL-4, rIL-5, and rIL-6 induced low or no expression of alpha mRNA of the patients' B cells. These results suggested that the patients lacked B cells able to produce transcripts for the IgA heavy chain, and that some patients' B cells might be defective at the switch-recombination process from mu to alpha or from mu and alpha to alpha.

Interleukin-2 reverses T cell unresponsiveness to Plasmodium falciparum-antigen in malaria immune subjects

Peripheral blood mononuclear cells (PBMC) from a large proportion of 34 healthy adult native residents in a malaria endemic area showed null or marginal proliferative response (low-responders) to schizont-enriched Plasmodium falciparum malaria antigen (M.Ag) but good response to pokeweed mitogen. In contrast, substantial proliferative response to M.Ag was observed in 8 8 adult temporary residents with a history of one to three acute malaria episodes. Purified CD4+ T cells preferentially responded to M.Ag, however in low-responders CD4+ T cell proliferation was poor. Moreover, no inhibition of CD4+ T cell proliferation was observed when graded numbers of CD8+ T cells were added in culture. The addition of recombinant interleukin 2 (rIL-2) to M.Ag restored the proliferative response of low-responders' PBMC. This response was M.Ag-specific when CD4+ T cells grown in M.Ag plus rIL-2, but not in rIL-2 alone, were tested in secondary cultures.

Immortalization of Epstein--Barr virus-infected CD23-negative B lymphocytes by the addition of B cell growth factor

Epstein-Barr (EB) virus-immortalized B lymphocytes coexpress the EB viral latent gene products (EB viral nuclear antigens 1 to 6, the latent membrane protein and the terminal protein gene products) and the cellular activation antigen CD23. Immortalized B cells can be separated from those which are infected but not immortalized on the basis of CD23 expression as early as 2 days after in vitro infection. In the present report we have confirmed these data, but show that if left in culture for 7 days after infection before separation the CD23-negative cells show a donor-related ability to become CD23-positive and immortalize. CD23-negative cells separated 2 days after infection can be induced to immortalize by the addition of low Mr B cell growth factor but not by the addition of recombinant interleukin 1, 4 or soluble CD23. At 2 to 3 days after infection the EB viral nuclear antigens 1, 2 and the high Mr species 3, 4 and 6, as well as the latent membrane protein can be detected in the CD23-positive fraction. In contrast at this time only nuclear antigens 1 and 2 could be detected in the CD23-negative fraction. This difference in gene expression may account for the inability of the CD23-negative fraction to immortalize. In the light of these observations the mechanism of viral persistence in vivo is discussed.

Dynamics of Immunoglobulin Synthesis After Major Trauma

Major mechanical trauma causes a severe suppression of B-cell differentiation and IgM synthesis in mononuclear leukocyte cultures. In this study the effect of recombinant lymphokines and physiologic T-cell supernatants on B-cell differentiation was investigated. The influence of potentially suppressing monocytes and positive regulatory T-lymphocytes was eliminated using purified B-cell cultures. Antigen-induced IgM synthesis was reduced on all days following trauma. Addition of recombinant interleukin 2 or T-cell supernatant enhanced but did not restore IgM synthesis. Although recombinant interleukin 4 and recombinant interferon gamma had a suppressive effect on IgM synthesis in controls, both lymphokines were ineffective in the patients' IgM synthesis. Spontaneous IgG production in patients was dramatically elevated, and the addition of lymphokines did not show any enhancing effect in patients. These results demonstrate that the IgM/IgG shift observed in peripheral blood mononuclear cell cultures also exists in purified B-cell cultures. Interleukin 2 partially restored suppressed IgM synthesis, while interleukin 4 and interferon gamma were ineffective in patients' B-cell differentiation. Interleukin 2 was the most effective lymphokine for the induction of Ig synthesis. These results lead us to conclude that the altered B-cell metabolism might also be responsible for the suppression of humoral immunity following trauma.

Direct activation of human B lymphocytes by Candida albicans-derived mannan antigen

We have studied the activation of human resting B cells by a carbohydrate antigen, mannan, with a polymannose branched repetitive structure. Mannan has been extracted from the cell wall of the Candida albicans yeast. For this purpose, dense G 0 B lymphocytes were purified from tonsils. Mannan antigen was shown to trigger B cell activation, since an increase of cell volume and RNA synthesis occurred. B cell proliferation was observed following addition of recombinant interleukin 2, but not following addition of recombinant interleukin 4 or low-molecular-weight BCGF. The B cell activation appears to be mannan-specific since B cells obtained from mannan-sensitized subjects but not from unsensitized subjects were responsive. The observation that mannan antigen can directly activate specific dense B lymphocytes can be related to the previous observation that the in vitro anti-mannan antibody production does not require a cognate T-B cell interaction.

In vitro and in vivo effects of interleukin 2 on the protozoan parasite leishmania.

T cells can have either resistance-promoting or disease-promoting effects in murine cutaneous leishmaniasis. It is known that the adoptive transfer of parasite-specific helper T cells led to an exacerbation of Leishmania-induced lesions. This work presents evidence that lymphokines produced by activated T cells could be involved in this exacerbating process by directly stimulating the parasite growth. In the presence of activated T cell supernatants, the in vitro growth of Leishmania mexicana amazonensis promastigotes was greatly enhanced. This effect was reproduced by addition of recombinant interleukin 2 (IL2). An anti-IL2 antibody partially reversed the stimulatory effect of IL. An in vivo in situ treatment of infected mice with IL 2 led to an exacerbation of the lesions. The increase in footpad swelling after IL2 treatment was correlated with a higher number of parasites per lesion. The protective effect of cyclosporin A against the development of Leishmania infection was abolished by IL2 treatment. As we observed that IL2 has a stimulatory effect on the in vitro Leishmania growth, we speculate that exacerbation of the lesions observed in vivo after IL2 treatment could be partially related with a direct effect of IL2 on the parasite growth.

T-Cell Colony Formation in Healthy Homosexual Men without Anti-Human Immunodeficiency Virus Antibody

In order to study the abnormal differentiation and proliferation of precursor T cells in homosexual men, a study was made of T-cell colony formation in 15 healthy homosexual men without anti-human immunodeficiency virus (HIV) antibody. The colony forming method used was the one-step monolayer technique using phytohemagglutinin as an inducer. Our results revealed that colony formation was extremely reduced and 3 cases showed no colony growth. However, with the addition of recombinant interleukin 2 to the culture system, the number of colonies increased up to the same level as in heterosexual controls. In addition, the analysis of CD3+, 4+, and 8+ cells of colony component cells revealed a decrease in CD3+ and 4+ cells. However, with the addition of recombinant interleukin 2, the numbers of CD3+ cells distinctly increased. The above results suggest that a disorder in the differentiation and proliferation of precursor T cells in homosexual men might be one of the causes of immunodeficiency.

Endotoxin Modulation of Hepatocyte Secretory and Cellular Protein Synthesis Is Mediated by Kupffer Cells

We hypothesize that alterations of hepatocyte function in sepsis are modulated by endotoxin (lipopolysaccharide)-triggered Kupffer cells. In the present experiments the effect of lipopolysaccharide on secreted and cellular proteins synthesized by hepatocytes cultured alone or cocultured with Kupffer cells was investigated using polyacrylamide gel electrophoresis. Lipopolysaccharide had no direct effect on the types or amounts of secreted proteins synthesized by hepatocytes alone. Kupffer cells coculturing resulted in increased synthesis of some hepatocyte proteins (68k and 23k) whose production was not altered by lipopolysaccharide. In contrast, addition of lipopolysaccharide to the hepatocyte-Kupffer cell coculture substantially decreased synthesis of several proteins (73k, 66k, and 35k). Despite an overall decrease in protein synthesis of hepatocytes cocultured with Kupffer cells after the addition of lipopolysaccharide, there was increased synthesis of several individual proteins (58k and 44k). Similar effects were seen in synthesis of cellular protein. The addition of recombinant interleukin 1 to hepatocytes alone or in coculture had no effect on the amount or type of protein synthesized. We conclude that Kupffer cells regulate the types and amounts of individual proteins synthesized by hepatocytes via mediators other than interleukin 1.

Responsiveness of T cells to mutant major histocompatibility complex class I antigen. I. Obligatory dependence of proliferative response on the presence of stimulator type accessory cells.

Proliferative response of splenic T cells of C57BL/6 mice to mutant major histocompatibility complex (MHC) class I antigen (H-2Kbm1) was examined with regard to the role of accessory cells. T cell proliferation in mixed lymphocyte culture (MLC) was not induced when accessory cells were removed from stimulator spleen cells by passage through Sephadex G-10 or nylon-wool column. Anti-Iab antibodies did not inhibit the proliferative response to class I antigen, whereas the same antibodies completely blocked the response to class II antigen (Iabm12). Accessory cells may not be mere presenters of MHC class I antigen because stimulator cells fixed with 0.05% paraformaldehyde lost the stimulating function. The proliferative response was partially recovered by the addition of recombinant interleukin 1 (IL-1) and/or IL-2 to MLC devoid of stimulator type accessory cells. It is concluded that stimulatory type accessory cells were obligatorily involved in the T cell proliferation, and the production of IL-1 by accessory cells is thought to play a critical role in this process.