CD4+CD25+ Regulatory T Cells Resist a Novel Form of CD28- and Fas-Dependent p53-Induced T Cell Apoptosis
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Ag receptor stimulation of preactivated T cells causes rapid cell death in an IL-2- and Fas-dependent manner. This phenomenon, known as activation-induced cell death (AICD), plays a pivotal role in the removal of Ag-reactive T cells after initial expansion. In this study, we report a novel form of T cell apoptosis that is distinct from classic AICD. When peripheral T cells were activated with anti-CD3 and anti-CD28 Abs precoated onto plastic plates, CD4(+)CD25(-) and CD8 T cells initially expanded but underwent massive apoptosis after 4 d. Unlike classic AICD, this type of T cell apoptosis pathway requires engagement of CD28 and expression of p53, a tumor-suppressor gene. The most striking feature of this form of apoptosis was regulatory T cell resistance. Under the same stimulating conditions, CD4(+)CD25(+) T cells grew continuously beyond 4 d. Consequently, when the entire CD4 population was cultured with plate-bound anti-CD3 plus anti-CD28 Ab, CD4(+)CD25(+)FoxP3(+) regulatory T cells outgrew nonregulatory T cells and expanded >7000-fold after 11 d. The data presented herein demonstrate a novel process of Ag-induced T cell death by sustained TCR and CD28 engagement and represent a simple and efficient procedure for the expansion of regulatory T cells in vitro.Keywords:
Regulatory T cell
Ag-presenting cells provide at least two distinct signals for T cell activation. T cell receptor-dependent stimulation is provided by presentation of a specific peptide Ag in association with MHC molecules. In addition, APC also supply costimulatory signals required for T cell activation that are neither Ag- nor MHC restricted. One such costimulatory signal is mediated via the interaction of B7 on APC with the CD28 receptor on T cells. Recently, CTLA-4 has been shown to be a second B7 receptor on T cells. In the present report, we have examined the expression of CD28 and CTLA-4 on a panel of resting and activated normal T cell subsets and T cell clones by RNA blot analysis in an attempt to determine whether their expression defines reciprocal or overlapping subsets. CD28 was detected in resting T cells, whereas CTLA-4 was not. After stimulation with PHA and PMA for 24 h, CTLA-4 mRNA was expressed in both the CD4+ and CD8+ subsets as well as in CD28+ T cells. We examined 37 human and six murine T cell clones that had been previously characterized for their cytokine production. After activation, CTLA-4 and CD28 mRNA were coexpressed in 36 of 37 human T cell clones and all six murine T cell clones. These included T cells of CD4+8-, CD4-8+, and CD4-8- phenotypes as well as clones with Th1 and Th2 cytokine profiles. In contrast, CD28 but not CTLA-4 mRNA was detected in leukemic T cell lines and myelomas. CTLA-4 and B7 mRNA but not CD28 mRNA was detected in two long term HTLV-I-transformed T cell lines. These data demonstrate that CD28 and CTLA-4 mRNA are coexpressed in most activated T cells and T cell clones, providing evidence that they do not define reciprocal subsets. Moreover, they are consistent with the hypothesis that B7 transmits its signal through a single receptor, CD28, on resting T cells, and multiple receptors, CD28 and CTLA-4, on activated T cells.
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Summary: In the present work, two important negative regulators of T cell responses in rats were examined. At the molecular level, rat CTLA-4, a receptor important for deactivating T cell responses, was examined for the expression pattern and in vitro functions. For this purpose, anti-rat CTLA-4 mAbs were generated. Consistent with the studies in mice and humans, rat CTLA-4 was detectable only in CD25+CD4+ regulatory T cells in unstimulated rats, and was upregulated in all activated T cells. Cross-linking rat CTLA-4 led to the deactivation of anti-TCR- and anti-CD28 stimulated (costimulation) T cell responses such as reduction in activation marker expression, proliferation, and cytokine IL-2 production. Although T cells stimulated with the superagonistic anti-CD28 antibody alone without TCR engagement also increased their CTLA-4 expression, a delayed kinetics of CTLA-4 upregulation was found in cells stimulated in this way. The physiological relevance of this finding needs further investigation. At the cellular level, rat CD25+CD4+ regulatory T cells were examined here in detail. Using rat anti-CTLA-4 mAbs, the phenotype of CD25+CD4+ regulatory T cells was investigated. Identical to the mouse and human Treg phenotype, rat CD25+CD4+ T cells constitutively expressed CTLA-4, were predominantly CD45RC low, and expressed high level of CD62L (L-selectin). CD25+CD4+ cells proliferated poorly and were unable to produce IL-2 upon engagement of the TCR and CD28. Furthermore, rat CD25+CD4+ cells produced high amounts of anti-inflammatory cytokine IL-10 upon stimulation. Importantly, freshly isolated CD25+CD4+ T cells from naive rats exhibited suppressor activities in the in vitro suppressor assays. In vitro, CD25+CD4+ regulatory T cells proliferated vigorously upon superagonistic anti-CD28 stimulation and became very potent suppressor cells. In vivo, a single injection of CD28 superagonist into rats induced transient accumulation and activation of CD25+CD4+ regulatory T cells. These findings suggest firstly that efficient expansion of CD25+CD4+ cells without losing their suppressive effects (even enhance their suppressive activities) can be achieved with the superagonistic anti- CD28 antibody in vitro. Secondly, the induction of disproportional expansion of CD25+CD4+ cells by a single injection of superagonistic anti-CD28 antibody in vivo implies that superagonistic anti-CD28 antibody may be a promising candidate in treating autoimmune diseases by causing a transient increase of activated CD25+CD4+ T cells and thus tipping ongoing autoimmune responses toward selftolerance.
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It has long been known that T cells require two signals for full activation, but the mechanisms of how these signals function have been only recently elucidated (1). The first signal is provided by the T-cell receptor after interacting with the MHC/antigenic peptide complex. This so-called “signal one” confers antigen specificity to the immune response but alone is insufficient for full T-cell activation. Indeed, T cells receiving only signal one are rendered anergic (unresponsive to antigenic rechallenge, with inhibition of proliferation and cytokine production) in vitro (2). The second signal, or “costimulatory signal,” is provided by interactions between specific receptors on the T cell and their respective ligands on antigen-presenting cells (APCs). The CD28/CD152–B7-1/B7-2 T-cell costimulatory pathway is a unique and complex pathway that regulates T-cell activation (recently reviewed in refs. 3 and 4) (Figure (Figure1).1). Interaction of CD28, constitutively expressed on T cells, with the B7 family of molecules (B7-1 and B7-2), expressed on APCs, provides a second “positive” signal that results in full T-cell activation, including cytokine production, clonal expansion, and prevention of anergy. In addition, CD28 signaling appears to be important in prevention of cell death and promotion of cell survival, presumably by upregulation of T-cell expression of bcl-xl genes (5).
Figure 1
Complexity of the CD28/CD152–B7-1/B7-2 T-cell costimulatory pathway. After antigenic stimulation (delivery of signal one through the T-cell receptor; not shown here), CD28, expressed on resting T cells, interacts with B7-2, and later with B7-1, ...
Once activated, T cells express another costimulatory molecule (CD152, or CTLA4) that is homologous to CD28, has a higher affinity to B7-1 and B7-2, and functions to provide a “negative” signal that inhibits cytokine production and arrests cell cycle progression (6–8). The importance of CTLA4 as a negative regulatory T-cell costimulatory molecule in the physiologic termination of T-cell responses (9) is highlighted by the observation that CTLA4 gene knockout mice develop massive lymphoproliferation and early death (10, 11). Furthermore, recent evidence suggests that CTLA4 negative signaling pathway may be required for the induction of acquired tolerance (12, 13). Indeed, it has been hypothesized that CTLA4 may function as a “master switch” for peripheral T-cell tolerance in vivo (14).
Several years before the regulatory function of CTLA4 was elucidated, Linsley et al. first described the creation of a new immunomodulatory agent that consists of the extracellular domain of the soluble CTLA4 receptor fused to the heavy chain of human IgG1 (6). Other similar agents have been subsequently described, including a murine form of CTLA4Ig, and several hundred articles have been published describing the immunomodulatory functions of CTLA4Ig in several experimental animal models of transplant rejection, autoimmunity, infections, asthma, and others (recently reviewed in refs. 3 and 4). Although it is clear that CTLA4Ig, because of the higher affinity of the CTLA4 receptor to B7-1 and B7-2, acts as a competitive inhibitor of CD28–B7-1/B7-2 costimulation and induces T-cell anergy in vitro, its exact mechanism of action in vivo remains unclear. It has been suggested, however, that induction of tolerance by B7 blockade may be due to anergy (failure of clonal expansion), deletion, or induction of regulatory T cells in vivo (15–21) (Figure (Figure1).1). Interestingly, a recent study from our group indicated that an intact CTLA4 negative signaling pathway is required for the immunosuppressive effects of CTLA4Ig in a mouse heart transplant model, adding further to the complexity of the B7-1/B7-2 costimulatory pathway in regulating immune responses (22).
After almost a decade of laboratory studies, CTLA4Ig finally “graduates” to the clinic. In this issue, Abrams et al. (23) present the results of a phase I clinical trial describing the immunosuppressive effects of CTLA4Ig in the T cell–mediated autoimmune skin disease psoriasis vulgaris. This study is unique because it is the first report describing the effects of blocking T-cell costimulatory activation in vivo in human disease. Moreover, although it is difficult to study mechanisms of action of new immunomodulatory therapies in humans, the authors describe the pathologic and immunologic correlates of CTLA4Ig therapy in their patient population. The study showcases the importance of continued T-cell activation in the pathogenesis of psoriasis. Furthermore, although efficacy results of phase I trials should be interpreted with extreme caution, it appears that CTLA4Ig is safe and is at least as effective as conventional therapy for psoriasis in a comparable patient population. However, what is most interesting is the potential for a prolonged beneficial clinical effect of therapy even after CTLA4Ig serum levels become undetectable. These cautionary data in particular suggest that CTLA4Ig may be inducing a state of T-cell hyporesponsiveness or tolerance in vivo.
Two interesting observations in this study again highlight the complexity of the CD28/CD152 T-cell costimulatory signaling pathway. First, there is the dichotomy between the clinical observation indicating that the beneficial effects of CTLA4Ig may be long lasting and the immunologic studies showing that fully primed T cell–dependent humoral immune responses were not affected, suggesting absence of immunologic tolerance. Second, there is the paradoxical result showing the divergence of suppression of cell-mediated and humoral immune responses at the high-dose schedule (50 mg/kg) of CTLA4Ig therapy. This latter observation and the recent studies by Judge et al. (22) make one wonder whether in certain diseases, complete blockade of B7-1/B7-2 may not be desirable because it may result in inhibition of a beneficial negative regulatory signal through CTLA4.
Although CTLA4Ig can now celebrate its graduation to the clinic, there is still much to learn. We need to understand in which diseases it is most effective and whether it provides a clear advantage over standard therapies (Table (Table1).1). We need to study its safety and efficacy profile in randomized, controlled trials. We need to determine what doses and protocols we should use in patients with different diseases. We need to explore whether CTLA4Ig may be used safely and effectively with other immunosuppressive agents or agents that block other costimulatory pathways, such as the CD40/CD154 pathway (24–26). Indeed, there are experimental data in small animals to indicate that calcineurin inhibitors, such as cyclosporine, may abrogate the immunosuppressive or tolerogenic effects of B7 and/or CD154 blockade (24, 27, 28). Finally, we need to investigate and better understand the exact mechanisms of costimulatory blockade in vivo in humans with different diseases and to develop surrogate markers to monitor disease activity and response to therapy.
Table 1
Human diseases in which CD28/B7 T-cell costimulatory blockade may have promise
In a recent study, Guinan et al. (29) used CTLA4Ig to anergize alloreactive bone marrow T cells ex vivo to prevent graft-versus-host disease after haploidentical bone marrow transplantation. Such studies, and the pioneering work by Abrams et al. (23), pave the way for the development of new clinical trials that will further examine the immunomodulatory functions of novel agents that block T-cell costimulatory activation in several immune-mediated human diseases. A better understanding of the mechanisms of these novel agents may make the goal of achieving immunologic tolerance in humans elusive no more.
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T cell activation requires co-engagement of the TCR with accessory and costimulatory molecules. However, the exact mechanism of costimulatory function is unknown. Mice lacking CD2 or CD28 show only mild deficits, demonstrating that neither protein is essential for T cell activation. In this paper we have generated mice lacking both CD2 and CD28. T cells from the double-deficient mice have a profound defect in activation by soluble anti-CD3 Ab and Ag, yet remain responsive to immobilized anti-CD3. This suggests that CD2 and CD28 may function together to facilitate interactions of the T cell and APC, allowing for efficient signal transduction through the TCR.
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Ag receptor stimulation of preactivated T cells causes rapid cell death in an IL-2- and Fas-dependent manner. This phenomenon, known as activation-induced cell death (AICD), plays a pivotal role in the removal of Ag-reactive T cells after initial expansion. In this study, we report a novel form of T cell apoptosis that is distinct from classic AICD. When peripheral T cells were activated with anti-CD3 and anti-CD28 Abs precoated onto plastic plates, CD4(+)CD25(-) and CD8 T cells initially expanded but underwent massive apoptosis after 4 d. Unlike classic AICD, this type of T cell apoptosis pathway requires engagement of CD28 and expression of p53, a tumor-suppressor gene. The most striking feature of this form of apoptosis was regulatory T cell resistance. Under the same stimulating conditions, CD4(+)CD25(+) T cells grew continuously beyond 4 d. Consequently, when the entire CD4 population was cultured with plate-bound anti-CD3 plus anti-CD28 Ab, CD4(+)CD25(+)FoxP3(+) regulatory T cells outgrew nonregulatory T cells and expanded >7000-fold after 11 d. The data presented herein demonstrate a novel process of Ag-induced T cell death by sustained TCR and CD28 engagement and represent a simple and efficient procedure for the expansion of regulatory T cells in vitro.
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Objective To study the expressions of CD25 in CD4+ T cells and CD28 in CD8+ T cells in the peripheral blood of HIV/AIDS. Methods CD25 in CD4+ T cells and CD28 in CD8+ T cells in the peripheral blood of 35 HIV/AIDS and 41 normal control were detected by flow cytometr. Results It shows that in patients with HIV/AIDS and the normal subjects,the expression percent of CD25 in CD4+T cells(27.51±4.23)%,(44.41±9.17)%、CD4+25+T cells(2.00±1.42)%,(16.62±4.60)%、CD4+25-T cells(5.16±3.37)%,(21.03±6.19)%、CD28 in CD8+T cells(25.12±6.33)%,(44.24±8.61)%, CD8+28-T cells(36.85±8.98)%,(13.33±4.58)%had significantly different (P0.01);CD8+28+T cells(12.31±4.14)%,(10.51±3.71)%had no significantly different (P0.05). Conclusions The low expression of CD25 in CD4+ T cells and CD28 in CD8+ T cells in HIV infection patients was closely associated with Immunodeficiency by HIV infection, the increase of CD8+28-T cells help to promote HIV/AIDS patients with inflammatory response and immune activation.
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T cells that play role in cell-mediated immunity must be activated in order to generate immune responses. The two signals are required for T cell activation. The first of these signals is provided by the antigen. The second signal is provided by the co-stimulatory molecules. For many years it was believed that T cells do not require co-stimulation for activation. However, in vitro and in vivo studies have shown that T cells need the co-stimulatory molecules for activation and expansion. Co-stimulation for T-cell activation and growth is provided by molecules of CD28 and the tumor necrosis factor (TNF) family members. Four co-stimulatory molecules belonging to the CD28 family have been identified. These family members are CD28, inducible co-stimulator (ICOS), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, CD152) and programmed cell death protein 1 (PD-1). Although CD28 and ICOS showed positive effects, CTLA- 4 and PD-1 molecule have a negative effect on T cell activation. In this review, we discussed co-stimulatory molecules belonging to the CD28 family, the ligands of these molecules, and the interactions of co-stimulatory molecules with ligands.
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