The accumulation of inflammatory cells in synovial sheath and epitenon during adhesion formation in healing rat flexor tendons.

The accumulation of inflammatory cells in synovial tissue was studied using indirect immunofluorescence assays on cell cultures and frozen tissue sections of healing rat digital flexor tendons. Flexor tendons were collected from rats 3, 7 and 14 days after crush injury. Tendon sheath and epithenon cells were isolated by sequential enzymic digestion and cultured for 2 days. Subpopulations of synovial and inflammatory cells were identified with MoAbs against cell surface glycoproteins present on B lymphocytes (CD45), T lymphocytes (CD2, CD4, CD8), macrophages (CD14) and endothelial cells. A phagocytosis assay was also used to identify macrophages. We report a substantial increase in the number of T lymphocytes (mainly helper/inducer) and phagocytotic cells with monocyte/macrophage surface markers in tendon sheath and epitenon 3 days after crush injury. The infiltration of inflammatory cells into synovial sheath and epitenon preceded an increase in fibronectin production by tendon cells which was seen 7 days after injury. To study the interaction between T lymphocytes and synovial cells in vitro, we established synovial fibroblast-like type B cell cultures and used stimulated and non-stimulated T lymphocytes in cell binding assays. We observed increased adhesiveness between unstimulated synovial cells and synovial cells previously cultured with activated and non-activated T lymphocytes. ELISA inhibition studies have shown an increase in fibronectin production by synovial fibroblasts co-cultured with stimulated CD4+ T lymphocytes. We suggest that the presence of inflammatory cells in synovial sheath and epitenon during tendon healing induces synovial fibroblasts and epitenon cells to increase their production of fibronectin, which provides a scaffold for subsequent adhesion formation.     

The effects of bone marrow stromal cell transplants on tendon healing in vitro

The purpose of this study was to investigate the effect of bone marrow stromal cells (BMSCs) on tendon healing in a canine ex vivo model. Bone marrow was harvested and BMSCs were isolated and cultured according to established protocols. Cells were seeded into 0.5 mg/ml collagen gels and cultured for 24 h to allow gel contraction, and then implanted between the lacerated ends of repaired flexor digitorum profundus tendons. Tendons repaired with a gel patch alone and without a gel patch served as control groups. After 2 and 4 weeks in culture, the repaired tendons were evaluated for breaking strength and stiffness. Cell viability was assessed by labeling the cells with PKH26 red fluorescent cell linker. The maximal strength and stiffness of repaired tendons with the BMSC-seeded patch were significantly higher than the repaired tendons without a patch or with a patch without cells, at both 2 and 4 weeks (p < 0.05). Viable BMSC were present between the cut tendon ends at both 2 and 4 weeks. We conclude that BMSC-seeded gel patch transplantation has the potential to enhance flexor tendon healing, and we plan to investigate this effect in vivo.     

The immunosuppressive effects of measles virus on T cell function--failure to affect IL-2 release or cytotoxic T cell activity in vitro.

Measles virus (MV) is known to depress T cell function. In order to determine whether this results from alteration in the production of, or response to, interleukin-2 (IL-2) we studied the effect of in vitro infection with MV on human IL-2 dependent T cell lines. MV produced a cytopathic productive infection in these cells. Class I allospecific cytotoxic T cells retained their cytotoxic activity 48 h after infection. Both cytotoxic and Leu 3a/4a positive T cell lines continued to respond to IL-2 by proliferation up to 26 h after infection. The ability of human tonsillar lymphocytes to generate IL-2 in response to phytohaemagglutinin following MV infection was then studied. In early measles infection (up to 48 h) there was no suppression of IL-2 production: in fact measles infected cells spontaneously released low levels of IL-2 in the absence of lectin. Similarly, IL-2 release was not affected by Herpes simplex virus infection of such cultures, although lymphocytes infected with Sendai or respiratory syncytial viruses produced considerably less IL-2. These observations suggest that MV-induced immunosuppression is not a result of inhibition of differentiated T cell function, IL-2 generation or responsiveness, but may be more directly related to virus-induced cytopathic effects in activated T cells.     

Regulation of human IgE response by T cells and their products

An in vitro experimental system for the induction of human IgE production has been established with human peripheral blood lymphocytes (PBL). For the assessment of IgE produced in vitro, a sensitive ELISA method has been developed by employing monoclonal anti-IgE antibodies. The stimulation of PBL with pokeweed mitogen plus Staphylococcus aureus strain Cowan I induced polyclonal IgE response. T cells from patients with pulmonary tuberculosis, after activation with purified protein derivative and/or IgE, suppressed selectively IgE response, and the suppressive activity was found to be mediated by IgE-specific suppressor factor with affinity for IgE molecules. The suppressive activity was effective both on spontaneous IgE production as well as on mitogen- or antigen-induced IgE response in the PBL culture from atopic patients. The affinity of the suppressor factor for IgE was suggested to be due to the structural similarity with Fc epsilon R on B cells. Absorption experiment suggest that the factor may bear class II major histocompatibility complex (MHC) molecules, although its effect was not MHC-restricted.     

Differential effects of estradiol and progesterone on human T cell activation in vitro

Estradiol (E2) and progesterone (P4) are steroid hormones important for the regulation of immune responses during pregnancy. Their increasing levels coincide with an improvement of T cell-mediated diseases such as multiple sclerosis (MS). Although immune-endocrine interactions are involved in this phenomenon, the relative contribution of hormones is not known. We here report a direct comparison of E2- and P4-mediated effects on human CD4⁺ T cells, key cells in immune regulation. T cells were stimulated to obtain different activation levels and exposed to a broad range of hormone concentrations. Activation level was assessed by CD69/CD25 expression by flow cytometry, and secreted proteins (n = 196) were measured in culture supernatants using proximity extension assay and electrochemiluminescence immunoassay. We found that in low activated cells, pregnancy-relevant E2 concentrations increased activation and the secretion of several immune- and inflammation-related proteins. P4, on the other hand, showed a biphasic pattern, where serum-related concentrations upregulated activation and protein secretion while placenta-relevant concentrations induced a prominent dampening irrespective of the initial activation level. Our results demonstrate the importance of P4 as a major hormone in the immune modulation of T cells during pregnancy and emphasize the need to further evaluate its potency in the treatment of diseases like MS.     

Tonic T cell signalling and T cell tolerance as opposite effects of self-recognition on dendritic cells

Naive T cells spend most of their time scanning the surface of dendritic cells (DCs), indicating that self-MHC/T cell receptor (TCR) interactions between these immune cells occur routinely in peripheral organs during the steady state. Peripheral self-MHC recognition on DCs drives seemingly opposing effects in the absence of inflammatory stimuli such as deletion of certain self-reactive T cells as well as maintenance of the T cell responsiveness to antigen, both of which shape the T cell repertoire and regulate T cell responses. Here we review recent data on the role of self-MHC recognition on steady-state DCs in the periphery and propose that interactions between T cells and steady-state DCs display an analogy with selection processes that occur in the thymus: high affinity TCR/self-MHC interactions in the periphery result in T cell deletion, while low/intermediate affinity interactions result in tonic TCR signalling that is required to keep T cells responsive to antigen.     

Discrimination of human CD4 T cell clones based on their reactivity with antigen-presenting T cells.

In this report, we describe the discrimination of human T cell clones based on their reactivity with activated T cells as antigen-presenting cells (APC). CD4+ T cell clones specific for peptide P30 of tetanus toxin (amino acids 947-967) and restricted to the DP4 molecule were established and tested for proliferation to peptide presented either by peripheral blood mononuclear cells (PBMC), Epstein-Barr virus (EBV)-transformed B cells or major histocompatibility complex (MHC) class II-expressing T cells. We found two sets of T cell clones: one set proliferated to peptide presentation by PBMC, EBV-transformed B cell lines (EBV-B cells) and MHC class II+ T cells (termed T-responder clones), while the other set of clones was only stimulated to proliferate, if the peptide was presented by PBMC or EBV-B cells, but not by T cells (T-nonresponder clones). Nevertheless, these T-nonresponder clones recognized P30 also on T cells, as revealed by Ca2+ influx. The discrimination of the clones was not due to different avidities of the T cell receptors (TcR) of individual clones for the MHC-peptide complex as T-responder and T-nonresponder clones had similar dose-response curves to P30 presented by fixed EBV-B cell lines. Addition of cytokines [interleukin (IL)-1, IL-2, IL-4 and interferon gamma] did not change the proliferative response of the clones, which was consistent throughout an observation period of greater than 4 months. T-nonresponder clones, exposed to P30 on MHC class II-expressing T cells, became not anergic, as they could be restimulated by P30 presented on EBV-B cells. The measurement of a panel of T cell activation markers and adhesion molecules on T-responder and T-nonresponder clones revealed a higher expression of the CD28 molecule on the T-nonresponder clones. The data suggest that freshly cloned T cells can be differentiated by peptide presentation on classical (PBMC, EBV-B cells) or non-classical APC (class II+ T cells), and that this discrimination is further underlined by different levels of adhesion molecules.     

The in vitro effects of metal cations on eukaryotic cell metabolism.

The in vitro cytotoxicity of nine metal cations common in dental casting alloys was evaluated using Balb/c 3T3 fibroblasts and four toxicity parameters: total protein production, 3H-leucine incorporation, 3H-thymidine incorporation, and MTT-formazan production. Concentrations causing 50% toxicity compared to controls (TC50's) and reversibility of these effects were determined. The range of potency of the metal cations was 2-3 orders of magnitude, with Cd2+ showing the greatest potency and In3+ showing the least. Potency did not correlate with atomic weight for these metals. For each metal cation, the TC50's of the various toxicity parameters were similar in most cases. However, several cations (Cu2+, Ga3+) showed greater potency with 3H-thymidine incorporation. Reversibility of the toxic effects was observed for all cations; the effects generally became irreversible at concentrations in the range of the TC50 value for each cation. Several stimulatory effects were seen. Small but statistically significant stimulations were observed after 24 h of metal exposure for Ag1+, Au4+, Cu2+, Ga3+, and Ni2+. Residual stimulations 24 h after removal of the metal cations were observed for Au4+, Cd2+, Ni2+, and Zn2+. Stimulations always occurred at concentrations below the TC50 concentrations. This study should be useful in evaluating the potential cytotoxic effects of metal cations released from dental alloys.     

Dose-response effects of 4-hydroperoxycyclophosphamide on human T and B cell function in vitro

4-Hydroperoxycyclophosphamide (4-OOH-CYP) is spontaneously converted in aqueous solution to 4-hydroxycyclophosphamide (4-OH-CYP), the major active metabolic of cyclophosphamide. We studied the dose related effects of in vitro treatment with 4-OOH-CYP on human T- and B cell-mediated immune responses. T-cell proliferation to mitogens and alloantigens was only partially inhibited even relatively high-doses of 4-OOH-CYP (greater than 6-12 micrograms/ml). In contrast cytotoxic functions of activated T-cells and natural killer (NK) cells were inhibited at lower doses (3-6 micrograms/ml). PWM induced in vitro synthesis of IgG by B-cells was inhibited at less than 3 micrograms/ml of 4-OOH-CYP. These data indicate that 4-HOO-CYP has selective, dose-dependent effects on human T and B cells in vitro.     

The effects of human cytomegalovirus challenge in vitro on subpopulations of T cells from seronegative donors

Purified T cells from seronegative donors were challenged with human cytomegalovirus (CMV) and assayed for response to the mitogen phytohemagglutinin (PHA) administered at various times after virus challenge. Cells were hyporesponsive to PHA added up to 6 h postinfection, while cells challenged with ultraviolet (UV)-inactivated CMV showed normal PHA responses. T-cell populations showed a complete inversion of helper/suppressor ratios by 6 h of infection, as determined by indirect immunofluorescence using the monoclonal antibodies OKT4 and OKT8. This increased proportion of suppressor cells was functionally demonstrable in an autologous one-way mixed lymphocyte reaction (AMLR).     

The in vivo and in vitro effects of caffeine on rat immune cells activities: B, T and NK cells

The effect of caffeine (naturally occurring plant methylxanthine) on immunological cell activities in Sprague-Dawley rat both in vivo and in vitro was studied. A cytotoxic assay was done to study natural killer (NK) cells and a proliferation assay was performed for T and B cell activities. Three different doses of caffeine i.e., 2, 6 and 18 mg/kg/day were administered chronically to Sprague-Dawley rats to assess the effects in vivo. Both NK cell cytotoxicity and B cell proliferative response to pokeweed mitogen (PWM) showed significant decreases (P less than 0.05) in rats treated with 6 mg/kg/day, whereas the T cell proliferative response to phytohemagglutinin-P (PHA-P) was significantly increased (P less than 0.05) in the rats treated with 18 mg/kg/day. In vitro, caffeine significantly decreases (P less than 0.05) B and T cell proliferative responses to PWM and PHA-P at added caffeine concentrations of 10, 20 and 40 micrograms/ml. However, no effect was observed on NK cells activity. Furthermore, in vitro, a broader dose range of caffeine (1, 10, 100 and 1,000 micrograms/ml) exhibited dose-dependent inhibition of both B and T cell proliferative responses.     

T-T cell interactions during in vitro cytotoxic T cell responses. VI. The role of T cell-derived colony-stimulating factor in helper T cell activation

A limiting dilution culture system has been used to analyze the role of T cell-derived colony stimulating factor (CSF) during the activation of IL 2-producing helper T lymphocytes (HTL). EL4 thymoma-derived supernatant (EL4-SN) increased about 4-fold the frequency of HTL precursors responding to metabolically active allogeneic stimulator cells. Upon biochemical separation this biological activity within the EL4-SN segregated from interleukin 2 (IL 2) and gamma interferon but was associated with or identical to CSF. Further, a comparable rise in HTL precursor frequencies was observed when semipurified interleukin 1 (IL 1) derived from the P388 D1 cell line was added to limiting dilution cultures. In contrast to IL 1, semipurified CSF failed to facilitate the activation of HTL precursors when heat-treated allogeneic spleen cells were used as stimulator cells. Because EL4-derived CSF was found to induce IL 1 production by macrophages we conclude that T cell-derived CSF amplifies, via stimulation of antigen-presenting cells the number of inducible HTL precursors. Therefore, the CSF/IL 1-dependent increase in HTL precursor frequencies reported here may reflect the differential activation threshold of HTL-precursors, most of which will not be activated by antigen per se but only in presence of additional cytokines.     

The effect of human placenta cytotrophoblast cells on the maturation and T cell stimulating ability of dendritic cells in vitro

The success of pregnancy depends upon regulatory mechanisms that allow the fetus to survive and develop to term in the uterus, despite maternal immune cells' awareness of paternal alloantigens. At least some of these specific mechanisms are mediated by the effect of fetal trophoblast cells. In the present study we examine the effect of human placental cytotrophoblast cells (CTCs) on the maturation of dendritic cells (DCs) in vitro. For that purpose, CTCs were isolated from samples of placentae at 5–11 weeks of gestation and co‐cultured with peripheral blood monocytes under conditions inducing DC maturation. CTC were shown to alter the morphology, phenotype and functional properties of DCs. As a result, a significant portion of cells acquire fibroblast‐like morphology and some of the cells retain the expression of CD14. DCs matured in the presence of CTCs do not differ from usual DCs in terms of CD80, CD83 and CD86 expression, as well as the ability to induce allogenic lymphocytes proliferation. However, CTCs reduce significantly the ability of DCs to stimulate interferon‐γ production and the loss of CD62L by T cells. The results obtained indicate that DCs may be involved in pregnancy‐associated changes of cytokine production and T cell migration.     

Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro

Embryonic stem cells (ESCs) have the potential to serve as a renewable source of transplantable tissue-specific stem cells. However, the molecular cues necessary to direct the differentiation of ESCs toward specific cell lineages remain obscure. Here we report the successful induction of ESC differentiation into mature functional T lymphocytes with a simple in vitro coculture system. The directed differentiation of ESCs into T cells required the engagement of Notch receptors by Delta-like 1 ligand (DL1) expressed on the OP9-DL1 stromal cell line. We found a normal program of T cell differentiation in ESC-OP9-DL1 cell cocultures. ESC-derived T cell progenitors effectively reconstituted the T cell compartment of immunodeficient mice, enabling an effective response to a viral infection. These findings provide a powerful tool for the molecular analysis of T cell development and open new avenues for the development of immunotherapeutic approaches using defined sources of stem cells.     

Early kinetics of B cell memory and the influence of T cells on the expression in vitro.

To study the early kinetics of B cell memory to sheep erythrocytes mouse spleen cells were cultured after short priming times in vivo. The influence of T cells on the expression of memory in vitro was investigated by treatment with anti-theta serum. Within 48 hours of in vivo priming part of the B cell population can differentiate into cells capable of IgG production after secondary antigen contact in vitro without the help of T cells. The presence of antigen was required for the in vitro development.     

The toxic effects of benzo[a]pyrene on activated mouse T cells in vitro

Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon contaminant that is widely present in environmental sources, including food. This study aims to clarify the effects of B[a]P toxicity on activated mouse T cells in vitro. Our results show that B[a]P markedly inhibited Concanavalin A (ConA)-induced T lymphocyte proliferation and suppressed the production of the cytokines Interferon (IFN)-γ, Interleukin (IL)-2 and IL-4. Western blot and protein-DNA interaction assays were used to study how B[a]P affects signal transduction. The results revealed that B[a]P suppressed the ConA-induced activation of the Ca²⁺/CaM/NFκB and Ca²⁺/CaM/CaN/NFAT signal transduction pathways. These observations indicate that B[a]P has toxic effects on activated mouse T cells in vitro.     

Characterization of Japanese éncephalitis virus-induced suppressor T cells and their products for delayed type hypersensitivity.

Intraperitoneal inoculation with Japanese encephalitis virus (JEV) induces the generation of T suppressor cells for delayed type hypersensitivity (Ts-DTH) in Swiss albino mice. The Ts-DTH are hydrocortisone resistant, partially sensitive to X-irradiation and the membrane phenotype of Ts cells is Ly I+. Ts-DTH suppression is mediated through the production of soluble suppressor factor (SF-DTH). SF-DTH is non-dialysable but passes through 450 nm filter and does not sediment after centrifugation at 100,000 g for 2 h; chromatography on Sephadex G-100 indicates an approximate molecular weight of 12,000.     

Action of deoxycoformycin on human T cell colonies in vitro.

Human peripheral blood mononuclear cells (HPBMC) from healthy individuals were stimulated polyclonally with pokeweed mitogen (PWM), in vitro, to produce IgG and IgM, which were subsequently measured by enzyme linked immunosorbent assay (ELISA). Indomethacin at 10(-6)-10(-8)M inhibited both IgG and IgM production but only when added within the first 24 hr of culture. PWM-stimulated HPBMC produced prostaglandin E2 (PGE2) during the first 24 hr of culture and this was completely inhibited by 10(-6)M indomethacin. Exogenous PGE2 added to PWM-stimulated HPBMC produced variable effects on immunoglobulin production at relatively high concentration (10(-6)-10(-7)M). However, reversal of inhibition of IgG production caused by 10(-7)M indomethacin was achieved with approximately 10(-7)M PGE2 but indomethacin-mediated inhibition of IgM production could not be fully reversed. No effect of either PGE2 or indomethacin could be detected on the PWM-induced proliferative response. These results suggest that prostaglandin E2, possibly with other arachidonic acid products, may be involved in the regulation of immunoglobulin production.