Production Of T-cell Growth Factor Research Articles

Cereblon is a novel E3 ubiquitin ligase regulator of CD28 signaling in T cells (P1007)

Abstract E3 ubiquitin ligases (E3-UbL) regulate clonal anergy and immune self-tolerance. Of the known regulators, c-Cbl and Cbl-b regulate T-cell growth factor production and proliferation induced by IL-2 through the CD28 pathway. Investigations of immunomodulatory (IMiDsTM) drugs lenalidomide (LEN) and thalidomide in myelodysplastic syndrome and multiple myeloma show that these analogs are capable of activating T-cells in the absence of co-stimulation and reverse T-cell anergy. LEN increased IL-2 production (p<0.001), proliferation (p<0.001), and pCREB promoter binding in primary human T-cells after TCR stimulation alone without CD28-receptor ligation. Comparing LEN-induced response in sorted human CD28+CD8+ and CD28-CD8+ T-cells and in CD28 shRNA knock-down T-cells indicates that LEN acts to directly potentiate receptor function dependent on CD28 expression. A landmark study identified cereblon (CRBN), a RING-domain E3-UbL as the primary target of thalidomide. CRBN has also been shown to mediate LEN immunomodulatory activity in vitro. To examine the effect of CRBN on CD28 signaling, germ-line CRBN knock-out mice (crbn-/-) were studied and found to have normal numbers of mature T-cells, normal naïve and memory compartmentalization, and normal proliferative response, but increased contextual activation of IL-2 in the absence of CD28 ligation. These results reveal a novel E3-UbL regulator involved in CD28 signaling with importance in anti-tumor immunity.

Development of a highly quantitative, reproducible assay for determination of chicken T cell growth factor biological activity

This report examines optimal culture conditions necessary for accurate and sensitive quantification of chicken T Cell Growth Factor (TCGF) activity. With this bioassay, TCGF is quantified by measuring its ability to cause proliferation of splenocytes prestimulated with mitogen. Proliferation is quantified by determining the optical density (OD) or “signal” of test samples in microtiter wells by measuring the incorporation of tetrazolium salt by live cells. To optimize assay conditions, systematic evaluation of the effects of cell culture variables was carried out with the constant aim of increasing signal to noise ratio in the assay. Higher signal to noise ratios were found when using Dulbecco's Modified Eagle's Medium (DMEM) rather than Roswell Park Memorial Institute Medium (RPMI) for basal tissue culture media containing the same supplements. The addition of lipid supplement to the assay system not only increased the proliferation signal, but also decreased the background OD. Incubation temperatures of 41 C rather than 37 C for both the mitogen prestimulation and proliferation phases of the assay also resulted in a higher signal to noise ratio. While incorporating the optimal experimental conditions, a finalized assay procedure employing test sample normalization with an internal assay standard was tested for accuracy. The assay can accurately detect 2 to 15 U/mL of TCGF activity. The within-assay variation ranged from 2 to 13% and the between-assay variation ranged from 11 to 22% depending upon the TCGF preparation being tested. The excellent reproducibility of this assay has facilitated investigations of TCGF production, processing, and purification.

Impairment of T-cell growth-promoting lymphokines in human insulin-dependent diabetes mellitus.

T-cell growth factor (TCGF) activity was studied in phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) from 10 type-1 diabetic patients who had been diagnosed within the previous 12 months (group A), from 9 diabetic patients in whom the duration of disease was more than 1 year (group B) and from 12 healthy controls (group C). The effects of indomethacin on PHA-induced TCGF activity and the effects of adherent cells (macrophages) from group A and group C on TCGF production of normal group-matched non-adherent cells (lymphocytes) were also studied. TCGF activity was assayed on TCGF-dependent blast cells and calculated as a stimulation index (SI). TCGF activity in group A (SI 0.86 +/- 0.8) was significantly different from that in group B (SI 1.75 +/- 1.02; P = 0.037) and in group C (SI 1.91 +/- 1.29; P = 0.023). Following the addition of indomethacin, TCGF SI was 1.35 +/- 0.74 in group A, 1.85 +/- 0.73 in group B and 2.06 +/- 1.19 in group C. The responses to indomethacin were found to correlate with the basal TCGF activity in all subjects (r = -0.48; P = 0.006) independently of the disease process studied or its duration. No correlation was found between TCGF activity and parameters of metabolic control (HBA1c and fructosamine). Interestingly, a significant inverse correlation was found between TCGF activity and the required dose of insulin only in group A (r = -0.66; P < 0.05). Adherent cells from diabetic patients were found not to inhibit TCGF production.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of physical exhaustion and glucocorticoids (dexamethasone) on T-cells of trained rats.

Following a previous observation that moderate physical training (running) of rats did not impair T-cells, in this study moderately trained Wistar rats were run to exhaustion on 2 consecutive days: in one case (T-dex) this was preceded by an intraperitoneal injection of 0.5 mg.kg-1 of dexamethasone (dex) and in the other case there was no prior injection (T). Similarly one group of sedentary control rats, was injected with dex (C-dex) and the other group was not (C). Rats were killed 24 h after the last treatment (dex, exercise). Compared with the C rats, the T rats exhibited a decreased number of thymocytes (75%), in particular CD4+CD8+ thymocytes and splenocytes (55%), notably CD4+CD8- splenocytes (P < 0.01). Also noted in the T rats was a lower (45%) in vitro (+mitogen) percentage of IL2r+CD4- splenocytes (expressing the IL2 receptor), and reduced (40%, P < 0.01) or unchanged in vitro production of T-cell growth factor (TCGF) by splenocytes or blood mononucleated cells (BMC), respectively. The dex decreased the number of thymocytes and splenocytes in the same way in T-dex rats (compared to T rats) and in C-dex rats (compared to C rats, P < 0.01). In T-dex rats compared with C-dex rats, on the other hand, dex had little effect on in vitro TCGF production by BMC, and no effect on other in vitro parameters. These results would indicate that physical exhaustion was responsible for an alteration in T-cells in the moderately trained rat. This alteration was in part enhanced by dex.

Effects of Chicken Anemia Virus on Cell-Mediated Immune Function in Chickens Exposed to the Virus by a Natural Route

Three-week-old chicks were inoculated orally with CAV and killed at various times postinoculation (PI). The spleens were removed, the cells were stimulated with concanavalin A, and lymphocyte transformation responses were determined. Supernatants from these cultures were also assayed for T-cell growth factor (TCGF) and interferon. Adherent macrophages from spleen or bone marrow were assayed for interleukin-1 production, Fc receptor expression, phagocytosis, and bactericidal activity. All CAV-inoculated chickens developed CAV antibodies, but no anemia was seen. Controls remained CAV-antibody-negative throughout the experiment. CAV-inoculated chickens showed significant differences from controls in their lymphocyte transformation responses and in production of TCGF and interferon. Differences were greatest at 14, 21, and 28 days PI. Significant differences were also observed in interleukin-1 production by spleen macrophages, as well as in Fc receptor expression, phagocytosis, and bactericidal activity of bone-marrow macrophages.

Immune system activation associated with a naturally occurring infection in Xenopus laevis

Immune system activation correlated with a naturally occurring infection has been found in the South African clawed frog Xenopus laevis. The microorganism thought to be the cause of this infection is coccobacilloid and approximately 1 μm in diameter. Since this microorganism does not grow on conventional bacterial media and it has been observed intracellularly, it may be an obligate intracellular bacterium. It has been found in Xenopus peripheral blood and in highly vascularized organs such as the spleen and liver. Splenomegaly is the only pathology thus far described for infected frogs; infection is not associated with increased morbidity or mortality. This infection has been found in all outbred frogs examined in shipments from one South African and three separate North American vendors, and has been transmitted to animals bred and raised in our laboratory. This infection has a profound effect on the immune system of Xenopus. Significant numbers of splenocytes from infected individuals exhibit morphology commonly associated with activated T lymphocytes. There is constitutive production of T-cell growth factor (TCGF) and both IgM and IgY. Freshly harvested splenocytes from infected animals proliferate in response to a TCGF-containing supernatant, indicating that they express receptors for TCGF, a trait exclusively exhibited by activated lymphocytes. These splenocytes also show an increase in the activation marker recognized by the monoclonal antibody FJ17.

Effect of moderate exercise on rat T-cells

The aim of this study was a detailed examination of the effects of moderate exercise on T-cells in adult male Wistar rats. The T-cell populations were compared in sedentary rats (C, n = 5) and in rats trained for 4 weeks on a treadmill (30-60 min.day-1, 6 days.week-1, 20-30 m.min-1) and sacrificed at rest (T-rest, n = 5). In the T-rest rats, there were higher percentages of CD4+CD8-, CD4-CD8+ and CD4-CD8- thymocytes (P < 0.05, P < 0.05 and P < 0.01 respectively) and of CD4-CD8+ splenocytes (P < 0.01), and a lower percentage of CD4-CD8+ cells in the lymph nodes (P < 0.01). Compared with T-rest or C rats, trained rats (n = 5) or untrained rats (n = 5) sacrificed immediately after a running session (60 min, 30 m.min-1) had a higher percentage of mononucleated cells CD4+CD8- in the blood (P < 0.05 and P < 0.01). Lastly, compared with C rats, rats (n = 5) sacrificed immediately after their 5th day of training (30-60 min.day-1) presented a higher total splenocyte population (P < 0.05) and greater in vitro production of T-cell growth factor (interleukin 2 + Interleukin 4) by splenocytes in response to a mitogen (P < 0.01). These results would indicate that moderate endurance training modifies the cellular composition of lymphoid organs, without impairing the in vitro functions of T-cells.

Effects of Chicken Anemia Agent on Lymphokine Production and Lymphocyte Transformation in Experimentally Infected Chickens

One-day-old chicks with no maternal antibodies to chicken anemia agent (CAA) were inoculated intramuscularly with CAA grown in MDCC-MSB1 cells. A control group of birds from the same source was inoculated intramuscularly with a lysate from uninfected MSB1 cells. Birds were killed at 8, 15, 22, 29, and 43 days postinoculation (PI), and the spleens were removed. Spleen cells were dispersed and stimulated with various concentrations of Concanavalin A (Con A), and lymphocyte transformation responses were determined. Supernatants from Con A-stimulated cultures were assayed for T-cell growth factor (TCGF) and interferon. Decreased lymphocyte transformation and TCGF production were demonstrated at 8 and 15 days PI. This was followed by a stimulation in activities before a return to control levels at 43 days PI. Interferon levels were elevated 8 days after infection. This was followed by a significant decrease in activity compared with controls at 15, 22, and 29 days PI, and a return to control levels by 43 days PI. The results suggest that CAA infection in young chickens can produce a dramatic decrease in immune competence, which, although transitory, is likely to seriously compromise the ability of birds to mount a successful immune response to invading pathogens.

Peripheral blood mononuclear cells in the squirrel monkey Saimiri sciureus II. — Exploration of the IL2/IL2R pathway of T-cell activation

We report here on functional aspects of squirrel moneky T cells isolated either from peripheral blood mononuclear cells or in the presence of accessory cells or accessory-cell-secreted cytokines. The T-cell response triggered by polyclonal activators was measured through proliferation, T-cell growth factor production and transient expression of receptors to growth factors. We also sought to delineate mechanisms by which antimitogenic molecules might prevent lymphocytes from being able to express “activation antigens” and/or from proliferating. Under these conditions, we mainly explored the inducible IL2/IL2R pathway and phenomena occurring in lymphocytes “non-activation” in terms of early and late cellular events; indeed, defects in the IL2/IL2R pathway represent one of the dysregulation events presumed to occur during the acute phase of human malaria, disease for which. Saimiri monkeys have been selected as experimental hosts.

Comparison of the immunosuppressive activities of the antimycotic agents itraconazole, fluconazole, ketoconazole and miconazole on human T-cells

Four antifungal agents have been screened in vitro for their immunosuppressive effects on proliferative responses in human mixed lymphocyte cultures (MLC). A hierarchy of inhibitory activity was observed, where itraconazole was > ketoconazole > miconazole > fluconazole, with itraconazole as suppressive as cyclosporin A, and fluconazole completely without suppressive activity. The mechanism of inhibition did not involve blockade of T-cell growth factor production and, consistent with this, interleukin-2-dependent T-cell clone proliferation was blocked by these agents in the same order of decreasing activity as in MLC. The secretion of cytokines without known T-cell growth factor activity (interferon-γ, tumour necrosis factor-α) was also not significantly blocked by these agents. These results therefore demonstrate that antifungal azole drugs may be variably strongly immunosuppressive for human T-lymphocyte proliferation in vitro, but none appear to be so via a mechanism involving inhibition of cytokine secretion.

Blastogenic responses and production of TCGF by mononuclear cells isolated from the bovine endometrium

Mononuclear cells (MNCs) were isolated from the endometrium of cattle by enzymatic digestion followed by centrifugation on a density gradient. Of the cells isolated, 32±3.2% rosetted AET-treated sheep red blood cells, 9±0.4% possessed surface membrane immunoglobulin, and 15±6.1% were esterase positive. The cells possessed the ability to proliferate in the presence of T cell mitogens and produced T cell growth factor (TCGF) after in vitro stimulation with Concanavalin A. Endometrial MNCs did not appear to suppress the blastogenesis of peripheral blood MNCs.

Interleukin-2 mRNA expression, lymphokine production and DNA synthesis in glutathione-depleted T cells

The stimulation of DNA synthesis in lymphocyte populations was previously shown to depend strongly on the intracellular glutathione (GSH) level. Since T cell growth is known to depend on interleukin 2 (IL-2), the experiments in this report were designed to determine whether intracellular GSH depletion may inhibit IL-2 production or the IL-2 dependent DNA synthesis. Our experiments revealed that IL-2 production and DNA synthesis of mitogenically stimulated splenic T cells have indeed different requirements for GSH. The addition of relatively high concentrations of GSH (5 m M) to cultures of concanavalin A (Con A)-stimulated splenic T cells was found to augment strongly the DNA synthesis but inhibited the production of IL-2. Moderate intracellular GSH levels, however, are apparently not inhibitory for IL-2 production, since intracellular GSH depletion by cysteine starvation or by graded concentrations of DL-buthionine sulfoximine (BSO) had virtually no effect on IL-2-specific mRNA expression and the production of T cell growth factor (TCGF). The DNA synthesis activity, in contrast, was strongly suppressed after GSH depletion with either method. As in cultures of splenic T cells, GSH depletion had no substantial effect on the induction of IL-2 mRNA and TCGF production in several mitogenically stimulated T cell clones. Taken together, our experiments suggest that complex immune responses may operate best at intermediate GSH levels that are not too high to inhibit IL-2 production but sufficient to support DNA synthesis.

Regulation of murine lymphokine production in vivo. III. The lymphoid tissue microenvironment exerts regulatory influences over T helper cell function.

We investigated the capacity of murine T lymphocytes, isolated from various lymphoid organs of normal or antigen-primed donors, to produce IL-2 or IL-4 after activation with anti-CD3 or specific antigen. Our results established that T cells resident within lymphoid organs being drained by nonmucosal tissue sites (e.g., axillary, inguinal, brachial lymph nodes, or spleen) produced IL-2 as the predominant T cell growth factor (TCGF) after activation. Conversely, activated T cells from lymphoid organs being drained by mucosal tissues (Peyer's patches, and cervical, periaortic, and parathymic lymph nodes) produced IL-4 as the major species of TCGF. Analysis of the lymphoid tissues obtained from adoptive recipients of antigen-primed lymphocytes provided by syngeneic donors provided evidence that direct influences were being exerted on T cells during their residence within defined lymphoid compartments. These lymphoid tissue influences appeared to be responsible for altering the potential of resident T cells to produce distinct species of TCGF. Steroid hormones, known transcriptional enhancers and repressors of specific cellular genes, were implicated in the controlling mechanisms over TCGF production. Glucocorticoids (GCs) were found to exert a systemic effect on all recirculating T cells, evidenced by a marked dominance in IL-4 production by T cells obtained from all lymphoid organs of GC-treated mice, or after a direct exposure of normal lymphoid cells to GCs in vitro before cellular activation with T cell mitogens. Further, the androgen steroid DHEA appeared to be responsible for providing an epigenetic influence to T cells trafficking through peripheral lymphoid organs. This steroid influence resulted in an enhanced potential for IL-2 secretion after activation. Anatomic compartmentalization of the DHEA-facilitated influence appears to be mediated by differential levels of DHEA-sulfatase in lymphoid tissues. DHEA-sulfatase is an enzyme capable of converting DHEA-sulfate (inactive) to the active hormone DHEA. We find very high activities of this enzyme isolated in murine macrophages. The implications of our findings to immunobiology are very great, and indicate that T cells, while clonally restricted for antigen peptide recognition, also appear to exhibit an extreme flexibility with regards to the species of lymphokines they produce after activation. Regulation of this highly conservative mechanism appears to be partially, if not exclusively, controlled by cellular influences being exerted by distinct species of steroid hormones, supplied in an endocrine or a paracrine manner where they mediate either systemic or tissue-localized influences, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of T-cell activation and T-cell growth factor (TCGF) production by hydrogen peroxide

Activated macrophages are known to release a variety of immunoregulatory substances including the low-molecular-weight substances hydrogen peroxide and lactate. We report here that lactate but not hydrogen peroxide is capable of supporting a substantial production of T-cell growth factor (TCGF) in cultures of accessory cell-depleted splenic T-cell populations after stimulation with concanavalin A. Hydrogen peroxide and its biosynthetic precursor superoxide anion (O − 2) mediate, however, a strong augmentation of the TCGF production by accessory cell-depleted T-cell populations in the presence of lactate. Lactate inhibits the incorporation of [ 3H]thymidine in short-term cultures (18–26 hr) of accessory cell-depleted T cells. This confirms the rule that (optimal) production of T-cell growth factor requires a growth inhibitory signal. Concentrations of hydrogen peroxide which augment TCGF production most effectively (i.e., 1 × 10 −5 M) do not inhibit the incorporation of [ 3H]thymidine; and higher concentrations (3 × 10 −5−1 × 10 −4 M) of hydrogen peroxide inhibit both the production of TCGF and the incorporation of [ 3H]thymidine. In agreement with the augmenting effect of hydrogen peroxide on TCGF production, it was observed that the proliferative response in mixed lymphocyte cultures is suppressed by catalase and augmented by 1 × 10 −5 M H 2O 2. Proliferative and cytotoxic responses in mixed lymphocyte cultures with an external source of interleukin 2 (IL-2) in contrast, are not augmented by 1 × 10 −5 M H 2O 2. The relatively high concentration of 1 × 10 −4 M hydrogen peroxide was found to inhibit the proliferative responses in mixed lymphocyte cultures with or without external IL-2 but not the cytotoxic response in the presence of IL-2. This indicates that CTL precursor cells may be relatively resistant against H 2O 2.

Regulation of T-cell functions by l-lactate

Lactate is a product of glycolytically active macrophages. After stimulation with concanavalin A accessory cell-depleted splenic T-cell populations were found to produce only minute amounts of T-cell growth factor (TCGF); but substantial amounts of TCGF were produced if the cultures were supplemented either with splenic adherent cells or with lactate but not with interleukin-1 (IL-1). IL-1 was capable, however, of supporting TCGF production by the thymoma subline EL4-6.1. TCGF production in cultures of accessory cell-depleted splenic T-cell populations was demonstrable with 10 −3 M l-lactate, and optimal responses (plateau level) were obtained with 4–6 × 10 −2 M l-lactate. Cultures of macrophages were found to accumulate up to 5 × 10 −2 M lactate. Our experiments indicate, therefore, that lactate serves as a regulatory signal by which macrophage-like accessory cells enhance helper-T-cell functions. Lactate is apparently not the only mediator of accessory cell function since plateau levels of TCGF production were markedly lower with lactate than with splenic accessory cells; but l-lactate was found also to determine the magnitude of T-cell-mediated immune responses in vivo and in cultures of unfractionated lymphocyte populations. The production of interferon in accessory cell-depleted and concanavalin A-treated T-cell cultures, however, was not significantly affected by lactate. Concanavalin A-stimulated splenic T-cell populations were found to consume glucose rapidly and to release lactate into the supernatant. This indicates that the cells contain more lactate and pyruvate than they can utilize by their respiratory metabolism. The administration of external lactate or pyruvate was found to inhibit the utilization of glucose by the mitogenically stimulated T cells.

Production and assay of ovine T cell growth factor by concanavalin A stimulated peripheral blood leukocytes

Conditions for the production and assay of ovine T cell growth factor (TCGF) activity were evaluated. Peripheral blood leukocytes were stimulated with concanavalin A (Con A) in the presence of 2% autologous serum or serum-free media. Supernatants were harvested after 30 hr and concentrated for further characterization. A 28 hr proliferation assay with 2.5 × 10 4 24 hr Con A blasts per well was optimal for detection of TCGF. Peak TCGF activity occurred with a 30–37KD molecular weight fraction. Production and assay of TCGF were performed under autologous conditions to reduce background stimulation which occurred when fetal bovine serum was present. This methodology required no cell lines or inbred animals and should be adaptable to the study of immunostimulatory molecules of other outbred species.

Induction of concanavalin A dose-dependent T-cell growth factor production by insertion of T-cell membrane components into the AKR thymic lymphoma BW 5147.

Sendai virus vesicles were used as vehicles for the insertion of various cell membranes into different cell lines. The transplantation efficiency was measured by using FITC-labelled concanavalin A (Con A) or monoclonal antibodies against the T-cell marker Lyt 2 and the major histocompatibility complex product H-2Db in a fluorescence-activated cell sorter. Results indicate that it is possible to transplant mitogen responsiveness to certain cell types. Con A stimulation of T-cell membrane transplanted BW 5147 showed that it is possible to induce a mitogen dose-dependent T-cell growth factor production. Consequently this method appears to be an attractive model for further study of the properties of the membrane structures involved in mitogen triggering of cells.

T lymphocytes, monocytes and erythropoiesis disorders in chronic renal failure.

The T lymphocytes, OKT4-depleted T lymphocytes, monocytes, macrophages and the bone marrow early erythroid progenitor (BFU-E) interactions have been evaluated in normal subjects and in uremic patients in predialysis phase (PDP), on hemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD). In the same subjects the T-cell growth factor (TCGF) activity and the T-cell subset identification have also been assessed. The results show in uremic patients, particularly in the presence of reduced OKT4 subset and TCGF activity, a blockage of the interactions between immunocompetent and bone marrow cells which normally could stimulate early erythroid progenitor growth. Since this blockage is partially removed by CAPD, this may suggest a more efficient removal of an interaction inhibitor with CAPD than with other dialysis techniques. The mechanism by which such a material may act appears unclear, but it is possible to suppose an inhibiting activity on the production of TCGF by lympho-monocyte cells which under normal conditions could be considered as an early erythropoiesis regulatory factor.

T-cell growth factor production by adult, but not childhood. Acute lymphoblastic leukaemic cells.

The production of T-cell growth factor (TCGF) by acute lymphoblastic leukaemia (ALL) cells was determined in seven children and in three adults. A significant production of TCGF by adult, but not childhood, ALL cells was observed. The adult ALL cells were classified as "non-T-non-B" by surface marker analysis. It is suggested that TCGF production may not be confined to the cells of T-lineage.

The differential effects of hydrocortisone on activation and tolerance induction in human T lymphocyte clones

The in vitro effects of hydrocortisone on T cell activation and tolerance induction were investigated using human influenza virus immune T cell lines and clones. Hydrocortisone at 10 −9 to 10 −6 molar concentrations was able to inhibit the antigen induced but not the T cell growth factor (TCGF) mediated proliferative response of both the lines and clones. However, hydrocortisone was able to inhibit TCGF production by cloned T cells. The proliferative response of cloned T cells to intact influenza virus A/Texas/1/77 was more markedly inhibited by equivalent concentrations of hydrocortisone than was the response of that clone to a 24 amino acid sequence (p20) of the haemagglutinin molecule implying that hydrocortisone may also act at the level of antigen processing. Furthermore hydrocortisone was able neither to induce T cell tolerance alone nor to inhibit antigen specific tolerance induction. However, hydrocortisone did lower the antigen threshold for tolerance induction. The possible mechanisms of hydrocortisone activity in the modulation of T cell regulation in autoimmune disease are discussed.