G1 arrest and high expression of cyclin kinase and apoptosis inhibitors in accumulated activated/memory phenotype CD4+ cells of older lupus mice

A general characteristic of lupus-prone mice (and humans) is the expedited accumulation of large numbers of presumably self-reactive activated/memory phenotype T cells. The mechanism by which these cells escape apoptosis has not been defined. We used activated/memory phenotype CD4⁺ cells from male BXSB mice with early-life severe lupus-like disease to investigate cell cycle status and apoptosis susceptibility, and to determine the role of corresponding genes in survival of these cells. In vitro acridine orange staining indicated that most of the rapidly accumulating memory phenotype CD4⁺ T cells of 4-month-old male BXSB mice are G1 arrested. Long-term bromodeoxyuridine in vivo labeling also showed that with advanced age, there was a shift of the CD4⁺ CD44^hi male cells from predominantly cycling to predominantly noncycling. Moreover, the CD4⁺ CD44^hi cells of older males were refractory to anti-CD3-induced proliferation and apoptosis. Using a multiprobe RNase protection assay encompassing riboprobe panels for cell cycle and apoptosis-related genes, we found that these cells exhibited high expression of certain members of the Ink4 (p18^Ink4C) and Cip/Kip (p21^Cip1) families of cyclin kinase inhibitors as well as of the apoptosis-inhibiting Bcl-x_L gene. Western blot analysis confirmed increased levels of Bcl-x_L and p21^Cip1, and also identified increases in another cyclin kinase inhibitor, p27^Kip1. We propose that in autoimmunity, self-reactive CD4⁺ cells are subjected to successive rounds of activation/division that eventually lead to a build-up in cyclin-dependent kinase inhibitors. Once high levels of such inhibitors are reached, they cause refractoriness to further activation, impaired cell cycle entry and resistance to apoptosis, a situation akin to replicative senescence.     

Preferential S Phase Entry and Apoptosis of CD4 T Lymphocytes of HIV-1-Infected Patients after in Vitro Cultivation

We have studied the relationship between spontaneous apoptosis and cell cycle perturbations in circulating peripheral blood lymphocytes of HIV-1-infected patients and healthy controls. PBMC obtained from HIV-1-infected patients and healthy controls were incubated in culture medium for 48 h. Cells were separated into CD4⁺ and CD8⁺ populations using immunomagnetic beads. Apoptosis and cell cycle phases were measured by propidium iodide staining and bromodeoxyuridine (BrdU) incorporation followed by flow cytometric analyses. In experiments using cells obtained from HIV-1-infected patients, spontaneous apoptosis was more frequent in CD4⁺ T lymphocytes than in CD8⁺ T lymphocytes (17.6% vs 9.5%, P < 0.005). Among healthy controls, spontaneous apoptosis in CD4⁺ and CD8⁺ T lymphocytes was comparable (4.5% vs 5.1%). Lymphocytes obtained from patients were more frequently in S phase than healthy controls' cells (2.2 ± 0.9% vs 0.5 ± 0.2%, P < 0.002) and patients' CD4⁺ cells tended to enter S phase more frequently than controls' CD4⁺ cells (4.2% ± 3.5% vs 1.8% ± 0.5% P < 0.04), whereas the frequency of S phase CD8⁺ T cells was not different among patients (2.8% ± 2.9%) and controls (1.8% ± 0.5%) (P > 0.4). Kinetic analyses using BrdU and PI staining revealed that S phase cells were more likely to become apoptotic than resting (G₀–G₁) cells (28.4% ± 10.3% vs 11.3% ± 9.9% in patients, P < 0.04, and 15.3% ± 2.8% vs 1.8% ± 0.5% in controls, P < 0.003). Lymphocytes obtained from HIV-1-infected persons are activated in vivo to enter S phase and to undergo spontaneous apoptosis after brief in vitro cultivation. The present studies indicate that most apoptotic cells in this system are CD4⁺ and kinetic analyses reveal that S phase cells are more likely to undergo spontaneous apoptosis than G₀–G₁ cells. Accelerated cell death in HIV-1 disease may contribute to the failure of lymphocyte responsiveness to appropriate T cell receptor stimulation.     

Prednisone increases apoptosis in in vitro activated human peripheral blood T lymphocytes

Glucocorticoid hormones (GCH) regulate, through the apoptotic process, the negative selection of immature T cells in the thymus. Because apoptosis seems to occur also in the maintenance of peripheral tolerance, we have investigated whether GCH may induce apoptosis in human mature lymphocytes. Peripheral blood lymphocytes (PBL) or peripheral CD4⁺ and CD8⁺ T cell subsets were cultured in the presence of phytohaemaglutinin (PHA) or PHA and prednisone (PDN) at 10⁻³-10⁻¹²M concentrations for 72, 96 and 120h. Cell cycle and membrane antigen expression were evaluated by flow cytometry and DNA degradation was detected by agarose gel electrophoresis. PDN blocks PBL growth in the G₁ phase of cell cycle and inhibits both IL-2 receptor (IL-2R) expression and IL-2 secretion. Apoptosis is clearly increased by PDN in PHA-activated human PBL, and the apoptotic effect of PDN is stronger on CD8⁺ than on CD4⁺ T lymphocytes. All these effects are dose- and time-dependent. The addition of exogenous IL-2 did not rescue lymphocytes from PDN-increased apoptosis. These results show that PDN increases apoptosis in mature activated human peripheral blood lymphocytes, suggesting a possible role of GCH in the maintenance of immune tolerance at post-thymic level.     

Constitutive expression of Bcl-xL or Bcl-2 prevents peptide antigen-induced T cell deletion but does not influence T cell homeostasis after a viral infection

We examined the CD8⁺ T cell response to lymphocytic choriomeningitis virus (LCMV) in mice doubly transgenic for an LCMV-specific TCR and for either bcl-x_L or bcl-2. Clonal down-sizing of the anti-viral CD8⁺ T cell response and the generation of T cell memory was not influenced by constitutive expression of these anti-apoptotic proteins in T cells. Expression of Bcl-x_L or Bcl-2 did, however, prevent LCMV peptide-induced peripheral deletion of mature CD8+ T cells in vivo and apoptosis of activated LCMV-specific effector T cells in vitro. The CD8⁺ T cells “rescued” by Bcl-x_L or Bcl-2 from peptide antigen-induced cell death were anergic and this could not be reversed by addition of IL-2 in vitro or by adoptive transfer into antigen-free recipient mice followed by LCMV infection in vivo. Taken together, we show here that 1) Bcl-x_L or Bcl-2 are functionally equivalent in their ability to modulate CD8⁺ T cell survival in vivo, 2) distinct apoptosis signaling pathways exist in CD8⁺ T cells, one that can be inhibited by Bcl-2 or Bcl-x_L and one that cannot be blocked, and 3) apoptosis of CD8⁺ effector T cells during the declining phase of an immune response is not prevented by constitutive expression of the anti-apoptotic proteins Bcl-x_L and Bcl-2.     

Role of tumour necrosis factor-alpha (TNF-α) in the induction of HIV-1 gp120-mediated CD4+ T cell anergy

The HIV-1 envelope glycoprotein (gp120) is known to induce antigen-specific and non-specific CD4⁺ T cell anergy. We found that early T cell activation, as indicated by HLA-DP expression in the early G₁ (G_1A) phase of the cell cycle, and the inhibition of mitogen-mediated IL-2 production induced by gp120, required TNF-α produced by gp120-stimulated macrophages. In the presence of an antibody to TNF-α, these changes induced by gp120 were inhibited, while recombinant TNF-α induced similar abnormalities of CD4⁺ T cells, even in the absence of gp120. On the other hand, inhibition of the mixed lymphocyte reaction (MLR) in CD4⁺ T cells by gp120, which may be related to gp120-mediated down-regulation of CD4 expression on T cells and activation of protein tyrosine kinase p56^lck in CD4⁺ T cells, was observed even in the absence of macrophage-derived TNF-α induced by gp120. These observations indicate that both TNF-α-dependent and independent events contribute to gp120-mediated CD4⁺ T cell anergy, and TNF-α appears to play an important role in inducing CD4⁺ T cell anergy in HIV-1 infection.     

TcR-α/β CD4CD8 T Cells in Humans with the Autoimmune Lymphoproliferative Syndrome Express a Novel CD45 Isoform That Is Analogous to Murine B220 and Represents a Marker of Altered O-Glycan Biosynthesis

Autoimmune lymphoproliferative syndrome (ALPS), caused by inherited defects in apoptosis secondary to mutations in genes encoding Fas/CD95/APO-1 and Fas ligand (Fasl)/CD95L, is characterized by nonmalignant lymphadenopathy and splenomegaly, increased T cell receptor α/β⁺ CD4⁻CD8⁻ T cells (α/β⁺ double-negative T cells [α/β⁺-DNT cells]), autoimmunity, hypergammaglobulinemia, and cytokine abnormalities. The α/β⁺-DNT cells are immunophenotypically and functionally similar to α/β⁺-DNT cells that accumulate in lpr and gld mice, which bear genetic mutations in Fas and FasL. In these mice, α/β⁺-DNT cells express the B-cell-specific CD45R isoform B220. We show that α/β⁺-DNT cells of ALPS patients, with either Fas or FasL mutations, also express B220. In addition, also similar to LPR/gLD mice, they have an unusual population of B220-positive CD4⁺ T cells. B220 expression, together with our finding of characteristic lectin binding profiles, demonstrates that cell surface O-linked glycoproteins have undergone specific modifications, which may have consequences for lymphocyte trafficking, cell–cell interactions, and access to alternative apoptosis pathways.     

Kinetics of Fas-Induced Apoptosis in Thymic Organ Culture

Although most thymocytes express high levels of Fas antigen (CD95), the role of Fas in apoptosis signaling during thymocyte maturation has not been defined. Fas apoptosis occurs primarily in the CD4⁺CD8⁺ subpopulations of thymocytes. Fas expression and apoptosis function were investigated in the CD4^–8^–, CD4⁺8⁺, and CD4⁺ and CD8 single positive thymocyte subpopulations by in vivo injection of anti-Fas and in vitro incubation of Fas with thymic organ cultures. Fas was first expressed on CD4^–8^– thymocytes coincident with expression of IL-2R and CD44. In Fas mutant lpr/lpr mice, defective Fas expression correlated with overproduction of late-stage CD4^–8^–-thymocytes. Fas was highly expressed on CD3^dull and CD3^bright thymocytes. CD4⁺8⁺CD3^dull thymocytes were sensitive to Fas apoptosis, whereas more mature CD4⁺8⁺CD3^bright thymocytes were resistant to Fas apoptosis. Anti-Fas incubation with established thymic organ culture for 24 hr resulted in apoptosis of approximately 25% of thymocytes. Continued incubation of thymic organ culture with anti-Fas resulted in an apoptosis rate of 25% of CD4⁺CD8⁺ thymocytes per day for the first 3 days of culture. Continued culture for further time points up to 6 days did not result in further apoptosis of the CD4⁺CD8⁺ thymocytes. These results suggest that CD4^–CD8^–CD44⁺ IL-2R⁺ thymocytes express Fas and there is overpopulation of the subsequent developmental stage of thymocytes in Fas mutant lpr mice. Also, early-stage CD4⁺8⁺ thymocytes are susceptible to Fas apoptosis, whereas Fas apoptosis resistance is required after 3 days of thymic organ culture. We conclude that these two populations of thymocytes are susceptible to Fas ligand-mediated apoptosis during T cell development in the thymus.     

Upregulated BclGL expression enhances apoptosis of peripheral blood CD4 T lymphocytes in patients with systemic lupus erythematosus

Increased lymphocyte apoptosis has been suggested to contribute to the development of systemic lupus erythematosus (SLE), but the critical factors involved in the apoptotic pathways are still unknown. By long serial analysis of gene expression (LongSAGE) profiles and microarray analyses, a novel apoptosis-related gene BclG_L expression was found significantly increased in peripheral blood CD4⁺ T cells of SLE patients, which was correlated with the enhanced CD4⁺ T cells apoptosis, anti-nuclear antibody (ANA) titer and proteinuria. In vitro, BclG_L expression could be specially upregulated by SLE serum stimulation and positively correlated with induced CD4⁺ T cell apoptosis. Enforcing BclG_L overexpression by lentivirus could directly enhance CD4⁺ T cell apoptosis, but these apoptosis-inducing effects could be partially inhibited by knockdown of BclG_L expression. Collectively, these results indicate that increased BclG_L expression may contribute to the aberrant CD4⁺ T cell apoptosis which causes an inappropriate immune response and impaired homeostasis in SLE.     

Developmental regulation of p53-dependent radiation-induced thymocyte apoptosis in mice

The production of T cell receptor αβ⁺ (TCRαβ⁺) T lymphocytes in the thymus is a tightly regulated process that can be monitored by the regulated expression of several surface molecules, including CD4, CD8, cKit, CD25 and the TCR itself, after TCR genes have been assembled from discrete V, D (for TCR-β) and J gene segments by a site-directed genetic recombination. Thymocyte differentiation is the result of a delicate balance between cell death and survival: developing thymocytes die unless they receive a positive signal to proceed to the next stage. This equilibrium is altered in response to various physiological or physical stresses such as ionizing radiation, which induces a massive p53-dependent apoptosis of CD4⁺CD8⁺ double-positive (DP) thymocytes. Interestingly, these cells are actively rearranging their TCR-α chain genes. To unravel an eventual link between V(D)J recombination activity and thymocyte radio-sensitivity, we analysed the dynamics of thymocyte apoptosis and regeneration following exposure of wild-type and p53-deficient mice to different doses of γ-radiation. p53-dependent radio-sensitivity was already found to be high in immature CD4⁻CD8⁻ (double-negative, DN) cKit⁺CD25⁺ thymocytes, where TCR-β gene rearrangement is initiated. However, TCR-αβ⁻CD8⁺ immature single-positive thymocytes, an actively cycling intermediate population between the DN and DP stages, are the most radio-sensitive cells in the thymus, even though their apoptosis is only partially p53-dependent. Within the DP population, TCR-αβ⁺ thymocytes that completed TCR-α gene recombination are more radio-resistant than their TCR-αβ⁻ progenitors. Finally, we found no correlation between p53 activation and thymocyte sensitivity to radiation-induced apoptosis.     

Mitochondrial perturbations define lymphocytes undergoing apoptotic depletion in vivo

We have recently shown that lymphocyte apoptosis induced by dexamethasone or superantigens is accompanied by reduction of mitochondrial transmembrane potential (ΔΨ_m) which precedes nuclear DNA fragmentation. Here, we demonstrate that fluorochromes such as 3,3′dihexyloxacarbocyanine iodide [DiOC₆(3)] which measure ΔΨ_m, or fluorochromes such as hydroethidine (HE) which measure mitochondrial superoxide anion production allow the identification of thymocytes or peripheral T lymphocytes which are eliminated by apoptosis in vivo. In mice bearing transgenic α/β T cell receptor (TCR) specific for a class I-restricted male-specific peptide, the superoxide-mediated oxidation of HE into ethidium (Eth) is enhanced among thymocytes which are being deleted due to negative selection (CD4⁺ CD8⁺ cells expressing the transgenic TCR in male mice) or lack of positive selection (CD4⁺ CD8^? thymocytes from female mice). ΔΨ_m reduction and/or enhanced HE oxidation are also found when apoptosis is induced by a series of pathogenic agents. Thus, lethal irradiation provokes mitochondrial and nuclear signs of apoptosis, and both these alterations are absent in mice bearing a p53 null mutation, underlying the correlation between mitochondrial perturbation and nuclear apoptosis. Similarly, superantigen-triggered deletion of peripheral T cells in vivo is accompanied by enhanced HE → Eth conversion and reduced DiOC₆(3) uptake. More importantly, as compared to normal controls. CD4⁺ or CD8⁺ cells from clinically asymptomatic human immunodeficiency virus-1 (HIV-1) carriers also contain a significantly elevated percentage of cells endowed with reduced DiOC₆(3) uptake. In superantigen- and HIV-induced apoptosis, the percentage of T lymphocytes with a subnormal DiOC₆(3) uptake is more important than that of cells marked by enhanced HE → Eth conversion. In conclusion, mitochondrial alterations precede and/or accompany nuclear signs of apoptosis induced by physiological and a variety of different pathogenic agents in vivo.     

Role of CD4+CD25+ Regulatory T Cells in Melatonin‐Mediated Inhibition of Murine Gastric Cancer Cell Growth In Vivo and In Vitro

Melatonin is an important immune modulator with antitumor functions, and increased CD4⁺CD25⁺ regulatory T cells (Tregs) have been observed in tumor tissues of patients and animal models with gastric cancer. However, the relationship between melatonin and Tregs remains unclear. To explore this potential connection, we performed an in vivo study by inoculating the murine foregastric carcinoma (MFC) cell line in mice and then treated them with different doses of melatonin (0, 25, 50, and 100 mg/kg, i.p.) for 1 week. The results showed that melatonin could reduce the tumor tissue and decrease Tregs numbers and Forkhead box p3 (Foxp3) expression in the tumor tissue. An in vitro study was also performed to test the effects of purified Tregs on melatonin‐mediated inhibition of MFC cells. The cell cultures were divided into three groups: 1) MFC+ Tregs; 2) MFC only; and 3) MFC+CD4⁺CD25^? T cells. After treatment with different concentrations of melatonin (0, 2, 4, 6, 8, and 10 mM) for 24 h, a dose‐dependent apoptosis and cell cycle arrest at the G2/M phase was detected in melatonin‐treated MFC at melatonin concentration higher than 4 mM. There were no significant differences in the rates of apoptosis and cell cycle distributions of MFC among the three groups. In conclusion, the antigastric cancer effect of melatonin is associated with downregulation of CD4⁺CD25⁺ Tregs and its Foxp3 expression in the tumor tissue. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

Selective infection of CD4 effector memory T lymphocytes leads to preferential depletion of memory T lymphocytes in R5 HIV-1-infected humanized NOD/SCID/IL-2Rγ mice

To investigate the events leading to the depletion of CD4⁺ T lymphocytes during long-term infection of human immunodeficiency virus type 1 (HIV-1), we infected human CD34⁺ cells-transplanted NOD/SCID/IL-2Rγ^null mice with CXCR4-tropic and CCR5-tropic HIV-1. CXCR4-tropic HIV-1-infected mice were quickly depleted of CD4⁺ thymocytes and both CD45RA⁺ naïve and CD45RA⁻ memory CD4⁺ T lymphocytes, while CCR5-tropic HIV-1-infected mice were preferentially depleted of CD45RA⁻ memory CD4⁺ T lymphocytes. Staining of HIV-1 p24 antigen revealed that CCR5-tropic HIV-1 preferentially infected effector memory T lymphocytes (T_EM) rather than central memory T lymphocytes. In addition, the majority of p24⁺ cells in CCR5-tropic HIV-1-infected mice were activated and in cycling phase. Taken together, our findings indicate that productive infection mainly takes place in the activated T_EM in cycling phase and further suggest that the predominant infection in T_EM would lead to the depletion of memory CD4⁺ T lymphocytes in CCR5-tropic HIV-1-infected mice.     

Cytokine-induced differentiation and proliferation of human T lymphocytesin vitro: Effects of interleukin 2 and interleukin 6

The contents of CD8⁺, CD4⁺CD8⁺, CD3⁺HLA-DR⁺, CD⁺INF-γ⁺ T cells, and natural killers (CD16⁺56⁺) and NK/T cells (CD16⁺56⁺CD3⁺) increase after 7-day culturing in the presence of interleukin-2. The number of apoptotic cells and cells in S-, and G₂+M phases of the cell cycle also increased. Interleukin-6 predominantly induced proliferation of CD3⁺HLA-DR⁺ T cells and G₂+M mitotic cells. Translated fromByulleten' Eksperimental'noi Biologii I Meditsiny, Vol. 129, No. 6, pp. 667–671, June, 2000     

CD8+ T cells mediate ultraviolet A-induced immunomodulation in a model of extracorporeal photochemotherapy

Extracorporeal photochemotherapy (ECP) that takes advantage of the immunomodulatory effects of UV light has been extensively used for many years for the treatment of several T cell–mediated diseases, including graft-versus-host disease (GvHD) and systemic scleroderma. Immune mechanisms that lead to the establishment of T cell tolerance in ECP-treated patients remain poorly known. In this study, we have tested the effect of UV/psoralen-treated BM-derived dendritic cells, referred to as ECP-BMDCs on the outcome of an antigen-specific T cell-mediated reaction, that is, contact hypersensitivity (CHS), which is mediated by CD8⁺ effector T cells (CD8⁺T_eff). The intravenous (i.v.) injection of antigen-pulsed ECP-BMDCs in recipient C57BL/6 mice induced specific CD8⁺ T cells endowed with immunomodulatory properties (referred to as CD8⁺T_ECP), which prevented the priming of CD8⁺T_eff and the development of CHS, independently of conventional CD4⁺ regulatory T cells. CD8⁺T_ECP mediated tolerance by inhibiting the migration and functions of skin DC and subsequently the priming of CD8⁺T_eff. CD8⁺T_ECP displayed none of the phenotypes of the usual CD8⁺T regulatory cells described so far. Our results reveal an underestimated participation of CD8⁺ T cells to ECP-induced immunomodulation that could explain the therapeutic effects of ECP in T cell-mediated diseases.     

Infection of Mice with Mycobacterium avium Primes CD8 Lymphocytes for Apoptosis upon Exposure to Macrophages

Mycobacterial infection is associated with granuloma formation in which the presence of apoptosis has been recognized. The role of CD4⁺ T and CD8⁺ T cells in host protection against mycobacterial infections has been demonstrated. Previous studies, however, have shown that CD8⁺ T cells have a limited role in host defense against Mycobacterium avium infection, and we hypothesize that M. avium infection could lead to T cell apoptosis. To investigate this hypothesis, C57BL/6 mice were infected with M. avium strain 101, and the rate of apoptosis of splenic lymphocytes cultured ex vivo with peritoneal macrophages was determined and compared with that of controls. When exposed to infected macrophages ex vivo, splenic lymphocytes from M. avium-infected mice underwent apoptosis, as determined by the TUNEL assay. This increased T cell apoptosis above the control level was observed after 3 weeks but not after only 1 week of infection in mice. No splenic T cell apoptosis was observed when lymphocytes from Mycobacterium smegmatis-infected mice were cultured in the presence of M. smegmatis-infected peritoneal macrophages. Likewise, macrophages infected in vitro with heat-killed M. avium did not trigger T cell apoptosis. Culture of macrophages in different chamber from lymphocytes, separated by a transwell membrane, was not associated with increase of apoptosis compared with uninfected control, suggesting a requirement for direct cell–cell interactions to trigger lymphocyte apoptosis. Using a double staining TUNEL followed by anti-mouse CD4 or anti-mouse CD8 monoclonal antibodies, it was observed that only CD8⁺ T cells but not CD4⁺ T cells underwent apoptosis at 3 weeks of infection. In conclusion, M. avium infection in C57/BL6 mice for 3 weeks renders CD8⁺ T cells prone to apoptosis when exposed ex vivo to macrophages infected with M. avium.     

Ionizing radiation induces apoptotic cell death in human TcR-γ/δ+ T and natural killer cells without detectable p53 protein

The p53 tumor suppressor gene has been shown to be involved in programmed cell death, apoptosis, in murine immature thymocytes after treatment with ionizing radiation. Ionizing radiation also induces apoptosis in peripheral mature lymphocytes. In this work, we investigated the p53 participation in radiation-induced apoptosis in human peripheral blood lymphocytes (PBL) subpopulations. Exposure to γ-irradiation resulted in an appreciable induction of apoptotic cell death in TcR-α/β⁺ (CD4⁺ and CD8⁺) T cells, TcR-γ/δ⁺ T cells, B cells and natural killer (NK) cells, as assessed by DNA fragmentation as well as the morphological characteristics. Importantly, it was found that there was a marked difference among PBL subpopulations as regards the induction of p53 protein by γ-irradiation. Similar to previous observations for murine thymocytes, p53 induction in TcR-α/β⁺ T cells and B cells after γ-irradiation was evident by Western blot analysis. Radiation-induced apoptosis in TcR-α/β⁺ T cells and B cells was efficiently inhibited by cycloheximide, indicating the requirement of de novo protein synthesis, including p53 protein, for radiation-induced apoptosis in both subpopulations. In marked contrast, no identifiable levels of p53 protein were induced in either TcR-γ/δ⁺ T or NK cells after γ-irradiation. In addition, it was demonstrated that radiation-induced cell death in TcR-γ/δ⁺ T and NK cells could be prevented by interleukin-2, but not by cycloheximide. These results imply that radiation-induced lymphocytic apoptosis can be mediated by p53-dependent or -independent mechanisms.     

The relative contribution of IL-4 and IL-13 to human IgE synthesis induced by activated CD4 or CD8 T cells

The relative contribution of IL-4 and IL-13 to the regulation of IgE synthesis has remained relatively poorly characterized, partially because of lack of suitable animal models. We have studied the roles of IL-4 and IL-13 in human IgE synthesis induced by supernatants derived from activated CD4⁺⁺ or CD8⁺ T cell clones. Neutralizing anti–IL-4 and anti–IL-13 monoclonal antibodies (mAbs) inhibited IgE synthesis induced by anti-CD40 mAbs and supernatants from CD4⁺ T cells by an average 61% and 42%, respectively (n = 25). Recombinant IL-13 had additive effects on IL-4-induced IgE synthesis, but only when IL-4 was present at low concentrations. Accordingly, IL-4 was the dominant IgE synthesis–inducing cytokine derived from highly polarized T helper (TH)₂ cells. However, anti–IL-13 mAbs also significantly inhibited IgE synthesis induced by two of three supernatants derived from allergen-specific T_H2-like cell lines generated from the skin of patients with atopic dermatitis. Furthermore, anti–IL-13 mAbs almost completely inhibited IgE synthesis induced by supernatants from T_H1 cells or CD8⁺ T cell clones. Taken together, these data indicate that IL-13, in addition to IL-4, contributes to IgE synthesis induced by all T helper cell subsets, including allergen-specific T_H2 cells. Moreover, IL-13 appears to be the major IgE synthesis–inducing cytokine derived from T_H1 cells or CD8⁺ T cells. (J Allergy Clin Immunol 1997;100:792-801.)     

Human renal tubular epithelial cells suppress alloreactive T cell proliferation

Renal tubular epithelial cells (TECs) are one of the main targets of alloreactive T cells during acute rejection. We hypothesize that TECs modulate the outcome of alloimmunity by executing immunosuppressive effects in order to dampen the local inflammation. We studied whether TECs possess immunosuppressive capacities and if indoleamine 2,3-dioxygenase (IDO) might play a role in suppressing T cell alloreactivity. Next, we studied the role of programmed death ligand 1 (PD-L1) and intercellular adhesion molecule-1 (ICAM-1 with regard to TEC-related immunomodulatory effects. CD3/CD28 and alloactivated peripheral blood mononuclear cells were co-cultured with activated TECs. We analysed CD4⁺ and CD8⁺ T cell proliferation and apoptosis in the absence or presence of IDO inhibitor 1-methyl-L-tryptophan (1-L-MT), anti-PD-L1 and anti-ICAM-1. Further, we examined whether inhibition of T cell proliferation was cell–cell contact-dependent. We found that TECs dose-dependently inhibited CD4⁺ and CD8⁺ T cell proliferation (P < 0·05). Activated TECs showed significantly increased IDO activity and up-regulated PD-L1 and ICAM-1 expression. Suppressed CD4⁺ and CD8⁺ T cell proliferation was only partially restored or failed to restore using 1-L-MT. Activated TECs increased early and late apoptosis of proliferating CD4⁺ and CD8⁺ T cells; only CD4⁺ T cell apoptosis was statistically affected by 1-L-MT. Transwell experiments revealed that TEC-mediated immunosuppression is cell–cell contact-dependent. We found that anti-ICAM-1 affected only CD4⁺ T cell apoptosis and not T cell proliferation. Our data show that TECs suppress both CD4⁺ and CD8⁺ T cell proliferation contact dependently. Interestingly, inhibition of proliferation and enhancement of apoptosis of T cell subsets is differentially regulated by indoleamine 2,3-dioxygenase and ICAM-1, with no evidence for the involvement of PD-L1 in our system.