Expression and co-stimulatory function of B7-2 on murine CD4+ T cells

Co-stimulatory signals mediated by the interaction of B7-1/B7-2 with CD28 are important for the activation of CD4⁺ T cells stimulated with antigen on antigen-presenting cells. There are controversies about the expression and function of B7-1/B7-2 on CD4⁺ T cells. The aim of this study was to analyze the expression of B7-1/B7-2 on naive and memory CD4⁺ T cells and the co-stimulatory function in the activation of naive CD4⁺ T cells stimulated by TCR ligation. Present results indicate that memory CD4⁺ T cells express B7-2 molecules on their surface, whereas naive CD4⁺ T cells do not. Neither memory nor naive CD4⁺ T cells expressed B7-1 molecule on their surface, although B7-1 mRNA was faintly expressed in memory T cells. B7-2 molecules expressed on memory T cells co-stimulated CD4⁺ naive T cells stimulated with plate-coated anti-CD3 to produce IL-2. Naive CD4⁺ T cells were shown to express B7-2 after co-stimulation with B7-2 and TCR ligation, because the naive T cells stimulated with anti-CD3 and B7-2CHO expressed B7-2 on their surface, although it remained to be studied whether the co-stimulation with B7-2 directly induced B7-2 expression on naive T cells. Our present results indicate that memory CD4⁺ T cells play some role in the activation of naive CD4⁺ T cells through the co-stimulation with B7-2 molecules.     

Human 4-1BB regulates CD28 co-stimulation to promote Th1 cell responses

Our present study provides evidence that the 4-1BB signal is critical to CD28 co-stimulation in maintaining T cell activation when CD28 has been down-regulated because of repeated stimulation. The 4-1BB signal synergized with CD28 co-stimulation by lowering the threshold of anti-CD28 required to sustain proliferation and IL-2 production. The 4-1BB signal also modulated CD28-mediated cytokine profiles by markedly enhancing Th1 but suppressing Th2-type cytokine production. The 4-1BB signal generated Th1-type cells, as identified by intracellular IFN-γ production. IFN-γ induction was detected preferentially in 4-1BB-expressing cells, but not in those expressing CD30. 4-1BB and CD30 were induced in both CD4⁺ and CD8⁺ cells, but the location of the two molecules was mutually exclusive in each T cell subset. Our study suggests that the 4-1BB signal regulates CD28 co-stimulation in the targeted subset cells to favor Th1 development and maintain long-term cell growth.     

Enhanced HIV expression during Th2-oriented responses explained by the opposite regulatory effect of IL-4 and IFN-γ on fusin/CXCR4

The human α-chemokine receptor fusin/CXCR4 is an important cofactor for entry of T lymphocyte-tropic HIV-1 strains. We investigated the possible regulatory role of T cell cytokine patterns on CXCR4 as well as HIV expression by using in vitro models of both secondary and primary immune responses. Antigen-specific memory CD4⁺ T cells infected with a T-tropic HIV-1 strain showed significantly higher CXCR4 and HIV-1 expression in Th0/2-oriented responses in comparison with Th1-oriented responses. Similarly, in naive CD4⁺ T cells activated in the presence of IL-4 or IL-12 and infected with the same T-tropic strain, IL-4 up-regulated whereas IL-12 down-regulated both CXCR4 and HIV-1 expression. The down-regulatory effect of IL-12 on CXCR4 expression was found to be dependent on its capacity to induce IFN-γ production. These observations can account for the higher risk of progression in HIV-1-infected individuals undergoing Th0/2-oriented immune responses.     

CD28 activation promotes Th2 subset differentiation by human CD4+ cells

Ligation of CD28 provides a costimulatory signal to T cells necessary for their activation resulting in increased interleukin (IL)-2 production in vitro, but its role in IL-4 and other cytokine production and functional differentiation of T helper (Th) cells remains uncertain. We studied the pattern of cytokine production by highly purified human adult and neonatal CD4⁺ T cells activated with anti-CD3, phorbol 12-myristate 13-acetate (PMA) and ionomycin, or phytohemagglutinin (PHA) in the presence or absence of anti-CD28 in repetitive stimulation-rest cycles. Initial stimulation of CD4⁺ cells with anti-CD3 (or the mitogens PHA or PMA+ionomycin) and anti-CD28 monoclonal antibodies induced IL-4, IL-5 and interferon-γ (IFN-γ) production and augmented IL-2 production (6- to 11-fold) compared to cells stimulated with anti-CD3 or mitogen alone. The anti-CD28-induced cytokine production corresponded with augmented IL-4 and IL-5 mRNA levels suggesting increased gene expression and/or mRNA stabilization. Most striking, however, was the progressively enhanced IL-4 and IL-5 production and diminished IL-2 and IFN-γ production with repetitive consecutive cycles of CD28 stimulation. The enhanced Th2-like response correlated with an increased frequency of IL-4-secreting cells; up to 70% of the cells produced IL-4 on the third round of stimulation compared to only 5% after the first stimulation as determined by ELISPOT. CD28 activation also promoted a Th2 response in naive neonatal CD4⁺ cells, indicating that Th cells are induced to express a Th2 response rather than preferential expansion of already established Th2-type cells. This CD28-mediated response was IL-4 independent, since enhanced IL-5 production with repetitive stimulation cycles was not affected in the presence of neutralizing anti-IL-4 antibodies. These results indicate that CD28 activation may play an important role in the differentiation of the Th2 subset in humans.     

Antigen presentation by interferon-γ-treated thyroid follicular cells inhibits interleukin-2 (IL-2) and supports IL-4 production by B7-dependent human T cells

The consequence of recognition of antigen on antigen-presenting cells that are induced to express major histocompatibility complex (MHC) class II molecules following an inflammatory process is still not clear. In this study, we have investigated the outcome of antigen presentation by epithelial cells and we have used as a model thyroid follicular cells (TFC) that are known to express MHC class II molecules in autoimmune thyroid diseases and acquire the capacity to present autoantigens to T cells infiltrating the thyroid gland. The result show that MHC class II-expressing TFC were unable to stimulate a primary T cell alloresponse, using CD4⁺ T cells from three HLA-mismatched responders. Phenotypic analysis showed that TFC, after incubation with interferon-γ, do not express the co-stimulatory molecules B7-1 (CD80) and -2 (CD86). Addition of murine DAP.3 cells expressing human B7-1 (DAP.3-B7) to cultures containing peripheral blood CD4⁺ T cells and DR1-expressing TFC led to a proliferative response, suggesting that the failure of TFC to stimulate a primary alloresponse was due to a lack of co-stimulation. Similarly, HLA-DR-restricted, influenza-specific T cell clones dependent on B7 for co-stimulation did not respond to peptide presented by TFC; again the lack of response could be overcome by co-culture of TFC with DAP.3-B7. Furthermore, recognition of antigen on TFC inhibited interleukin-2 (IL-2) production in the B7-dependent T cells. In contrast, in T helper type 0 (Th0) T cells, IL-4 release was not affected by TFC presentation. In addition, antigen presentation by TFC favored IL-4 production relative to IL-2 production by B7-indpendent Th0 clones. These results suggest that antigen presentation by MHC class II⁺ TFC may induce tolerance in autoreactive Th1 cells but may simultaneously favors a Th2 response in uncommitted T cells, and thereby support autoantibody production.     

Interleukin-4 and -10 exacerbate candidiasis in mice

Neutralization of endogenous interleukin (IL)-4 or IL-10 in mice with Candida albicans infection initiates or accelerates development of a T helper (Th)1-associated protective response. Here, we report the effect of IL-4 and IL-10 administration on the course of systemic or gastrointestinal (GI) candidiasis and on the development of Th immunity using yeast/host combinations that result either in Th1-associated self-limiting infection (healer mice) or in Th2-associated progressive disease (nonhealer mice). Treatment with IL-4 or IL-10 greatly exacerbated the course of systemic infection in nonhealer mice and rendered healer mice, inoculated with attenuated yeast cells, susceptible to infection. Under the latter conditions of yeast challenge and IL-4/IL-10 administration, the development of a fatal disease was associated with inhibition of IL-12 production and detection of progressive Th2 cell dominance. In contrast, in healer mice allowed to resolve their infections and to develop long-lived anti-candidal resistance, the expression of this acquired resistance was not impaired by IL-4 and/or IL-10, as shown by the outcome of reinfection with virulent yeast cells. In the GI model of infection, both IL-4 and IL-10 were found to exacerbate the course of infection and to induce the appearance of CD4⁺ T cells producing high levels of IL-4 and IL-10 in Peyer's patches. These findings demonstrate that exogenous IL-4 and IL-10 may greatly affect the development of Th responses to C. albicans in vivo, but do not modify the expression of established and predominant Th1 cell reactivity.     

Differential response of CD4+ V7+ and CD4+ V7− T cells to T cell receptor-dependent signals: CD4+ V7+ T cells are co-stimulation independent and anti-V7 antibody blocks the induction of anergy by bacterial superantigen

V7 is a novel cell surface glycoprotein that is expressed on 25% of circulating T lymphocytes. This molecule appears to play a critical role in T cell activation based on the observation that a monoclonal anti-V7 antibody inhibits T cell receptor (TCR)-dependent interleukin-2 (IL-2) production and proliferation of T cells. In the current study, CD4⁺ V7+ and CD4⁺ V7? T cells were separated from one another and their response to various stimuli analyzed. Although there were only minor differences between the two subsets in the expression of activation/differentiation markers, including CD45RA and R0 isotypes, when exposed to immobilized anti-CD3 or anti-TCR antibodies in the absence of APC, CD4⁺ V7+ T cells alone produced IL-2 and proliferated vigorously. By contrast, CD4⁺ V7? cells responded poorly to such stimuli, but they recovered their capacity to respond if antigen-presenting cells (APC) or anti-CD28 antibody were added to the cultures. The enhancement of the V7? T cell response by APC appears to be related to augmentation of TCR signals because the effect could be blocked by antibodies against molecules on APC [major histocompatibility (MHC) class II, CD86] that are known to up-regulate such signals through their interaction with counter-receptors on T cells. To assess the role of V7 in a system independent of co-stimulation, CD4⁺ T cells were stimulated with the bacterial superantigens, staphylococcal enterotoxins A and B. The cells responded by proliferating and then becoming anergic. Addition of anti-V7 antibody at the initiation of culture with superantigen did not inhibit cellular proliferation but prevented T cells from becoming anergic, while addition of anti-CD28 antibody had no effect on either proliferation or anergy induction. These results indicate that V7 and CD28 mediate distinct intracellular signals and suggest that V7 functions to preserve T cell reactivity whether the stimulus is mitogenic or anergizing.     

The outer surface lipoprotein OspA of Borrelia burgdorferi provides co-stimulatory signals to normal human peripheral CD4+ and CD8+ T lymphocytes

Studies in man and mice have indicated that T cells induced during Borrelia burgdorferi infection are involved in the pathogenesis of the disease. We analyzed the ability of B. burgdorferi to provide co-stimulatory signals to highly enriched normal human CD2⁺ T lymphocytes in the presence of suboptimal concentrations of immobilized anti-CD3 antibodies. Here we show that the lipid-containing recombinant outer surface lipoprotein A (rlip-OspA) of B. burgdorferi but not its delipidated derivative rNS1-OspA augmented CD3-induced T cell proliferation in a dose-dependent manner and at levels similar to that obtained with anti-CD28 antibodies. Lipopolysaccharide had no effect in this system at any concentration tested, suggesting that the active principle of co-stimulation is associated with the lipid moiety of rlip-OspA and distinct from conventional lipid A. Furthermore, incubation of CD2⁺ T cells or selected CD4⁺ as well as CD8⁺ subpopulations with rlip-OspA, but not with rNS1-OspA led to the production of interferon (IFN)-γ, interleukin (IL)-6 and tumor necrosis factor (TNF)-α, but not IL-4. In contrast, co-stimulation of the respective T cell populations with anti-CD28 antibodies resulted in the generation of IFN-γ, IL-4 and TNF-α, but not IL-6. This indicated that the signal transduction pathway induced by rlip-OspA is distinct from that elicited via the CD28 receptor. Co-stimulation of T cells with rlip-OspA also resulted in the development of cytolytic effector cells. In light of the fact that inflamed tissues of B. burgdorferi-infected hosts contain blood leukocytes together with spirochetes, their degradation products, or both, these results suggest that infiltrating CD4⁺ and CD8⁺ T cells of any specificities, including spirochetes, autoantigens, or both, participate in the pathogenesis of Lyme disease.     

Endogenous retroviral superantigen presentation by B cells induces the development of type 1 CD4+ T helper lymphocytes

The endogenous retroviral superantigen, minor lymphocyte stimulating antigen (Mls 1^a, encoded by Mtv-7), when presented by highly purified B cells induced the development of a highly polarized population of T helper (Th)1 cells from naive peripheral CD4⁺ T cells in vitro. Immobilized anti-Vβb6⁺ antibodies similarly generated highly polarized, largely Vβ6⁺, Th 1 populations in vitro. In the presence of exogenous interleukin-4, both stimuli were capable of generating Th 2, rather than Th 1 populations. Mls 1^a presentation by B cells in vivo led to the development of an equally polarized Th 1 population. Using monoclonal antibodies against interferon-γ and transforming growth factor-β it was demonstrated that maximal Th 1 development with either stimulus in vitro was dependent on the endogenous production of these two cytokines. Thus, our results demonstrate that the retroviral encoded superantigen, Mls 1^a, drives the development of Th 1 cells both in vitro and in vivo, and they suggest that B cell presentation does not, in itself, lead to the generation of Th 2 cells.     

Acute stimulation generates Tim‐3‐expressing T helper type 1 CD4 T cells that persist in vivo and show enhanced effector function

T‐cell immunoglobulin and mucin domain 3 (Tim‐3) is a surface receptor expressed by T helper type 1 (Th1) effector CD4 T cells, which are critical for defence against intracellular pathogens and have been implicated in autoimmune disease. Previous studies showed that Tim‐3 expression makes Th1 cells more susceptible to apoptosis and also marks functionally impaired T cells that arise due to chronic stimulation. However, other studies suggested that Tim‐3‐expressing Th1 cells do not always have these properties. To further define the relationship between Tim‐3 and Th1 cell function, we analysed the characteristics of Th1 cells that expressed Tim‐3 in response to brief stimulation in vitro or an acute viral infection in vivo. As expected, cultured CD4 T cells began expressing Tim‐3 during Th1 differentiation and secondary stimulation generated Tim‐3^? and Tim‐3⁺ fractions that were separated and further analysed. When injected into naive mice, Tim‐3⁺ cells down‐regulated Tim‐3 and survived equally well compared with Tim‐3^‐ cells. Further, Tim‐3^? and Tim‐3⁺ Th1 cells had similar functional responses when transferred into naive mice that were subsequently infected with lymphocytic choriomeningitis virus (LCMV). Cultured Th1 cells that expressed Tim‐3 following T‐cell receptor stimulation had a greater capacity to express signature Th1 cytokines than their Tim‐3^? counterparts and showed differential expression of genes that regulate CD4 T‐cell function. Consistent with these findings, Tim‐3⁺ Th1 cells generated in response to LCMV infection displayed augmented effector function relative to Tim‐3^? cells. These results suggest that Tim‐3 expression by Th1 cells responding to acute stimulation can mark cells that are functionally competent and have an augmented ability to produce cytokines.     

B7-1-dependent co-stimulation results in qualitatively and quantitatively different responses by CD4+ and CD8+ T cells

To characterize better the co-stimulatory activity of native B7-1 in the absence of other receptor/ligand interactions that might contribute to the response, B7-1 was purified by monoclonal antibody (mAb) affinity chromatography. Immobilization of purified B7-1 with anti-T cell receptor (TCR) mAb on cell-sized latex microspheres provided an effective stimulus for activation of both CD4⁺ and CD8⁺ T cells as measured by proliferation, development of effector function, and changes in motility and adhesion. The CD4⁺ T cell response was prolonged and resulted in efficient interleukin-2 production and clonal expansion. In contrast, CD8⁺ responses were transient. Proliferation and clonal expansion peaked on days 3 and 4, coincident with maximal expression of lytic effector function, and the cells then died. These results demonstrate that B7-1 mediated co-stimulation is sufficient for the induction of effector function in both helper and cytotoxic T cell precursors, but suggest that B7-1 co-stimulation is not sufficient to sustain helper-independent CD8⁺ CTL responses. When the dose responses of CD4⁺ and CD8⁺ T cells to B7-1 were compared, CD8⁺ T cells were found to require higher densities of B7-1 to attain an equivalent level of activation, suggesting that the level of expression of B7-1 by APC may influence the development of helper or CTL responses. Finally, in contrast to results obtained by others with B7-1 transfectants, purified B7-1 did not provide co-stimulation when presented on a surface separate from the TCR stimulus.     

CD28 ligands CD80 (B7-1) and CD86 (B7-2) induce long-term autocrine growth of CD4+ T cells and induce similar patterns of cytokine secretion in vitro

The interaction of CD28 and its ligands is critical for antigen-inducedT cell activation. Recent studies have demonstrated the existenceof at least two members of the B7 receptor family. In this report,the co-stimulatory signals provided by CD80 (B7-1) or CD86 (B7-2)were compared to CD28 ligation by mAb. We demonstrate that thekinetics of induction of T cell proliferation after anti-CD3stimulation was similar regardless of the form of co-stimulation.Similarly, B7-1 and B7-2 could both maintain long-term expansionof CD4 cells. The co-stimulatory effects of both B7-1 and B7-2were dependent on CD28 cross-linking, based on complete inhibitionof proliferation by CD28 antibody Fab fragments. Co-stimulationwith B7-1 and B7-2 induced high levels of cytokine secretionby resting T cells, and the effects of B7-1 and B7-2 could notbe distinguished. This conclusion is based on analysis of theinitial activation of CD28⁺ T cells. as well as T cell subpopulationsconsisting of CD4⁺ and CD8⁺ T cells. Both B7-1 and B7-2 couldelicit IL-4 secretion from CD4⁺ T cells while anti-CD28 antibodyinduced substantially less IL-4 secretion. Furthermore, bothB7-1 and B7-2 could stimulate high levels of IFN- and IL-4 fromCD4⁺CD45RO⁺ cells, while neither B7 receptor could co-stimulateIFN- and IL-4 secretion from CD4⁺CD45RA⁺ T cells. B7-1 and B7-2could, however, co-stimulate CD4⁺CD45RA⁺ T cells to secreteIL-2. By contrast, when previously activated T cells were tested,re-stimulation of CD4⁺ T cell blasts with B7-1 or B7-2 resultedin higher secretion of IL-4 and IL-5 than anti-CD28, while re-stimulationwith anti-CD28 antibody maintained a higher level of secretionof IL-2 and IFN- than B7-1 or B7-2. These observations may haveimportant implications because they suggest that the mannerof CD28 ligation can be a critical determinant in the developmentof cytokine secretion that corresponds to T_h1- and T_h2-likepatterns of differentiation. Together these observations suggestthat there are no Intrinsic differences between B7-1 and B7-2in their ability to co-stimulate the populations of cells thatwe have tested.     

Bach2 in CD4+ T cells from SLE patients modulates B-cell differentiation and IgG production

T and B cells participate in the development of systemic lupus erythematosus (SLE). BTB and CNC homology 2 (Bach2) is an irreplaceable regulator in the T and B lineages that helps to maintain immune homeostasis. However, the function of Bach2 in the pathogenesis of SLE has not been studied in depth. Flow cytometry and qRT‒PCR were used to assess Bach2 levels, bisulfite sequencing PCR was used to measure the methylation level, and silencing by electroporation and stimulation with a cytokine concentration gradient were used to investigate the effect of Bach2 on T cells. Bach2 expression was elevated in the helper T-cell subsets (T follicular helper, Th1, Th2, Th17, and Treg cells) of SLE patients and negatively correlated with disease severity and autoantibody levels. CD4⁺ T cells from SLE patients had decreased methylation levels in the Bach2 promoter region. Silencing Bach2 in CD4⁺ T cells induced increases in the CD19⁺ B-cell count, plasmablasts, and secretion of IgG by prompting the secretion of cytokines. The activation signals CD3/CD28, IL-6, and IL-21 upregulated Bach2 expression in CD4⁺ T cells. The regulation of Bach2 by cytokines and T-cell activation signals in CD4⁺ T cells was shown to act on B cells and play a protective role against SLE.     

Human recombinant interferon-β influences T helper subset differentiation by regulating cytokine secretion pattern and expression of homing receptors

Type I interferons (IFN) are important regulators of both innate and acquired immunity. We have used an in vitro system of human CD4⁺ T cell differentiation to determine how IFN-β influences development of T helper (Th) subsets and homing receptor expression. IFN-β promoted differentiation of CD4⁺ T cells that produce low levels of both IFN-γ and lymphotoxin compared to interleukin (IL)-12-derived Th1 CD4⁺ T cells. IFN-β inhibited production of Th2 cytokines (IL-5 and IL-13) and augmented IL-12-mediated IL-10 secretion. In addition, IFN-β significantly enhanced L-selectin expression on CD4⁺ T cells and synergized with IL-12 to induce expression of cutaneous lymphocyte-associated antigen (CLA). This Th1 L-selectin⁺, CLA⁺ phenotype is characteristic of T cells found in normal human skin and suggests a role for type I IFN in the regulation of Th subset differentiation and tissue-specific homing receptors.     

Prostaglandin E2 primes naive T cells for the production of anti-inflammatory cytokines

In addition to their capacity to induce pain, vasodilatation and fever, prostaglandins E (PGE) exert anti-inflammatory activities by inhibiting the release of pro-inflammatory cytokines by macrophages and T cells, and by increasing interleukin (IL)-10 production by macrophages. We here report that PGE₂, the major arachidonic acid metabolite released by antigen-presenting cells (APC), primes naive human T cells for enhanced production of anti-inflammatory cytokines and inhibition of pro-inflammatory cytokines. Unfractionated as well as CD45RO^?CD31⁺ sort-purified neonatal CD4 T cells acquire the capacity to produce a large spectrum of cytokines after priming with anti-CD3 and anti-CD28 monoclonal antibodies (mAb), in the absence of both APC and exogenous cytokines. PGE₂ primes naive T cells in a dose-dependent fashion for production of high levels of IL-4, IL-10 and IL-13, and very low levels of IL-2, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and TNF-β. PGE₂ does not significantly increase IL-4 production in priming cultures, whereas it suppresses IL-2 and IFN-γ. Addition of a neutralizing mAb to IL-4 receptor in primary cultures, supplemented or not with PGE₂, prevents the development of IL-4-producing cells but does not abolish the effects of PGE₂ on IL-10 and IL-13 as well as T helper (Th)1-associated cytokines. Addition of exogenous IL-2 in primary cultures does not alter the effects of PGE₂ on naive T cell maturation. Thus PGE₂ does not act by increasing IL-4 production in priming cultures, and its effects are partly IL-4 independent and largely IL-2 independent. Together with the recent demonstration that PGE₂ suppresses IL-12 production, our results strongly suggest that this endogenously produced molecule may play a significant role in Th subset development and that its stable analogs may be considered for the treatment of Th1-mediated inflammatory diseases.